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DETAILED STRUCTURAL ASPECTS OP THE

HERPES SIMPLEX VIRUS GENOME

Andrew John D a v iso n

A T h e s is p r e s e n te d f o r th e D egree o f

D o cto r o f P h ilo s o p h y

IN

The P a c u lty o f S c ie n c e

a t th e U n iv e r s i t y o f G lasgow

I n s t i t u t e o f V ir o lo g y S ep tem b er, 1^81Church S t r e e t G lasgow G il 5JH S c o t la n d

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TABLE OP CONTENTS

AC KNOWLEDGEMENTS

SUMMARY

ABBREVIATIONS

PART A: INTRODUCTION 1 -3 7

THE HERPESVIRUSES 1

C l a s s i f i c a t i o n and p a t h o g e n ic i t y 1

S tr u c tu r e o f th e v ir io n 4

S tr u c tu r e o f th e HSY genome 6

S tr u c tu r e s o f o th e r h e r p e s v ir u s genom es 10

THE LYTIC CYCLE OP HERPES SIMPLEX VIRUS 14

I n t e r a c t io n o f v ir u s w ith th e c e l l 14

DNA s y n t h e s is 19

RNA s y n t h e s is 24

P r o te in s y n t h e s is and m o d if ic a t io n 29

Enzyme a c t i v i t i e s in d u ced by HSV 34

PART B; MATERIALS AND METHODS 3 8 -7 0

MATERIALS 38

V ir u s e s 38

C e l l s 38

B a c te r ia 38

T is s u e c u ltu r e m edia and s o lu t i o n s 38

R a d io ch em ica ls 39

C h em ica ls 40

DNA 41

Enzymes 41

O ther m a te r ia ls 42

Commonly used s o lu t io n s and b u f fe r s 42

METHODS 4 3

Growth o f c e l l s 43

P r o d u c t io n o f v ir u s s to c it s 43

V ir u s t i t r a t i o n 43

P la q u e p u r i f i c a t i o n o f HSV 44

G en era tio n o f iiS V -l/ilS V -2 i n t e r t y p i c 44recom b in an t v i r u s e s , .

P r e p a r a tio n o f ilSV DI\1 A 45

P r e p a r a t io n o f PRV DNA 47■>

P r e p a r a t io n o f b a c te r io p h a g e lam bda DNA 48

E s t im a t io n o f DNA c o n c e n tr a t io n s 49

P u r i f i c a t io n o f r e s t r i c t i o n e n d o n u c le a se s 49

R e s t r ic t io n e n d o n u c lea se d ig e s t io n 51

A garose g e l e l e c t r o p h o r e s i s 51

P o ly a c r y la m id e g e l e l e c t r o p h o r e s i s 52

G en era l l a b e l l i n g o f DNA w ith *^P 52

I s o l a t i o n o f r e s t r i c t i o n fr a g m e n ts 54

T r a n s fe r o f DNA fra g m en ts to n i t r o c e l l u l o s e 55

B lo t h y b r id is a t io n 55

A u torad iograp hy 57

R e s t r ic t io n fragm en t n o m en cla tu re 57

C a lc u la t io n o f r e l a t i v e m o la r i t i e s 58o f r e s t r i c t i o n fra g m en ts

R a t io n a le f o r mapping r e s t r i c t i o n s i t e s 58

C on tam in ation o f r e s t r i c t i o n fra g m en ts 59i s o l a t e d from g e l s

G en era tio n o f b a c t e r ia l c lo n e s b e a r in g 60recom b in an t p lasim lds w ith riSV DNA i n s e r t s

32L a b e l l in g o f DNA te r m in i w ith P 64

D e t e r m i n a t i o n o f t h e n u c l e o t i d e b7s e q u e n c e o f DuA

PART C: RESULTS AND DISCUSSION

SECTION 1 : MAPPING OP RESTRICTION SITES ON IISV-1 ANN HSV-2 DNA

R e s u lt s

I n tr o d u e t io n

M o lecu la r w e ig h t c a l i b r a t i o n

R e la t iv e m o la r i t i e s o f r e s t r i c t i o n fra g m en ts

IISV-1 Kpnl p h y s ic a l map: r e c le a v a g e d a ta

HSV-1 Kpnl p h y s ic a l map: d o u b le d i g e s t d a ta

HSV-1 Kpnl p h y s ic a l map: h y b r id is a t io n d a ta

IISV-1 Kpnl p h y s ic a l map: r e l a t i v e o r d e r o f fra g m en ts n and p

HSV-l Kpnl p h y s ic a l map

P h y s ic a l maps f o r o th e r r e s t r i c t i o n e n d o n u c le a se s

D is c u s s io n

SECTION 2 : HOMOLOGY BETWEEN HERPESVIRUS GENOMES

R e s u lt s

I n tr o d u c t io n

D is t r ib u t io n o f hom ologous se q u e n c es betw een HSV-1 and HSV—2 DNA

C o l in e a r i t y o f th e genomes o f HSV-1 and HSV-2

N o n -c o lin e a r hom ology betw een th e genom es o f IISV-1 and IISV-2

Homology betw een th e HSV genome and the genom es o f BHV-1 and PRV

Homology betw een th e HSV and CMV genom es

S tu d ie s o f re c o m b in a tio n betw een th e HSV-1 and ilSV-2 genom es

P is c u s s io n

f e a t u r e s o f n u c le ic a c id h y b r id is a t io n

C o l in e a r i t y o f th e genom es o f HSV-1 and HSV-2

C onserved r e g io n s in th e genom es o f HSV-1, HSV-2, EHV-1 and PRV

Homology betw een th e genom es o f HSV and HCMV

D is t r ib u t io n o f r e g io n s hom ologous to ]JSV DNA in th e genom es o f EHV- 1 and PRV

N o n -c o l in e a r hom ology

G e n e tic r e la t e d n e s s o f h e r p e s v ir u s e s

Homology and r e co m b in a tio n i n HSV

D oes o n ly one HSV genome arrangem ent p a r t i c ip a t e in . reco m b in a tio n ?

SECTION 3 : NUCLEOTIDE SEQUENCES OP THE JOINT BETWEEN THE- L AND S SEGMENTS OP THE GENOMES OP. HSV-1 AJp HSV-2

R e s u lt s


S s t I r e s t r i c t i o n maps o f th e HSV-1 and HSV-2 j o i n t r e g io n s

O ther r e s t r i c t i o n maps o f th e HSV-1 and IISV-2 j o i n t r e g io n s

S iz e v a r i a b i l i t y i n th e j o i n t r e g io n

Homology betw een th e j o in t r e g io n s o f o f HSV-1 and HSV-2 DNA

1P in e r e s t r i c t i o n maps o f th e j o in t r e g io n s o f HSV-1 and HSV-2

L o c a t io n o f th e v a r ia b le r e g io n w ith in th e HSV-1 a seq u en ce

D e f i n i t i o n o f th e genom ic te r m in i and l o c a t i o n o f th e a seq u en ce

Tandem r e i t e r a t i o n s i n th e j o i n t r e g io n

C om parison o f n u c le o t id e seq u en c es

E x p r e ss io n o f th e IISV-1 j o in t r e g io n

gage

92

92

94

95

98

99

100

101 103

103

107

107

107

107

109

112114

115

115

116

118

120

121

Page

D is c u s s io n 124

F e a tu r e s o f r e i t e r a t i o n s 124

C oding p o t e n t i a l a t th e 3 ' end o f 125Vmw IE 175 mRNA

R o le o f th e a seq u en ce in c o d in g f o r 126p o ly p e p t id e

Homology betw een th e j o in t r e g io n s o f 127th e ilSV-1 and HSV-2 genom es

R o le o f th e a seq u en ce i n m a tu r a tio n o f 127genome le n g t h DNA

R ole o f th e a seq u en ce i n segm ent in v e r s io n 128

SECTION 4 : INVERSION OP THE TWO SEGMENTS 130OP-THE HSV GENOME . . .

R e s u l t s 130

I n tr o d u c t io n 130

S tr u c tu r e s o f th e j o i n t s and te r m in i 131o f B x l(2 8 -1 )

G ross s t r u c t u r e s o f E x l ( 2 8 - l ) s u b c lo n e s 133

S t r u c tu r e s o f th e j o i n t s and te r m in i o f 134B x l( 2 8 -1 ) su b c lo n e s

S t r u c tu r e s o f th e j o i n t s and te r m in i 138p f o t h e r reco m b in a n ts

E x p r e s s io n o f IE 1 1 0 /1 1 8 by B x l(2 8 -1 ) 140s u b c lo n e s

S t r u c tu r e s o f s u b c lo n e s o f B x l(2 8 -1 ) 142s u b c lo n e s

S tr u c tu r e o f th e genome o f RE4 142

D is c u s s io n 147

P ix e d and f r e e reco m b in a n ts 147

M odels f o r segm ent in v e r s io n 149

M echanism s f o r genome c i r c u l a r i s a t i o n 151and m a tu ra tio n

E x p r e s s io n o f B x l(2 8 -1 ) s u b c lo n e s 154d e le t e d in TR^/IR^

Genome s tr u c tu r e o f RE4 155

REFERENCES

AC KN Q \ i LEDGem eN TS

I sh o u ld l iK e to thank P ro f e s s o r John H. Subak-Sharpe

f o r £ )r o v is io n o f l a c i l i t i e s in th e I n s t i t u t e o f V ir o lo g y and

f o r h i s o v e r a l l s u p e r v is io n o f and en co u ra g in g i n t e r e s t in

t h i s work. I am g r e a t ly in d e b te d to Dr N e i l M. W ilk ie f o r

h i s g en ero u s a d v ic e and g u id a n ce th ro u g h o u t t h i s work and

f o r v a lu a b le c r i t i c i s m o f th e m a n u sc r ip t . I sh o u ld a l s o

l i k e to th an k Dr H .3 . lviarsden and Dr F .J . R ixon f o r t h e ir

c o l la b o r a t io n , Dr J.M . W halley f o r m aking a v a i la b le EHV-1

r e s t r i c t i o n en d o n u c lea se maps p r io r to p u b l i c a t io n , Mrs

M. Murphy f o r i s o l a t i n g s u b c lo n e s o f B x l ( 2 8 - 1 ) , and th e

many members o f th e I n s t i t u t e who p ro v id ed much a d v ic e and

s t im u la t in g d i s c u s s io n . The s e r v i c e s p ro v id e d by th e

Y/ashroom, S to r e , and M edia and C y to lo g y D epartm ents were

much a p p r e c ia te d .

D u rin g t h i s work th e a u th o r was em ployed by th e

M ed ica l R esea rch C o u n c il and, e x c e p t where o th e r w is e

s p e c i f i e d , a l l th e r e s u l t s r e p o r te d were o b ta in e d by th e

a u th o r ’ s own e f f o r t s .

JU. M.ull'

Xuj. o LiiCo-i-o i c o 'C i i ue 3 Lne a o r i v a t i o n o i r o 3 i r i c Lion

e n d o n u c l e a s e maps l o r n3V-1 and nSV—2 HnA; a s t u d y o l t h e

h o m o lo g o u s r e g i o n s oe tw een t h e genomes o f IiSV-1, HSV-2, EHV-1

and PRV, and Hie e f f e c t o f ho o o l o g y upon r e c o m b i n a t i o n

b e tw e e n HSV-1 ana . .3V-2; n u c l e o t i d e s e q u e n c e s 01 t n e L-S

j o i n t r e g i o n s i n HSvr- l and 113V-2 Hi.A; and an a n a l y s i s o f two

HSV—l/H SV -2 i n t e r t.ypic r e c o m b i n a n t s w h ich f a i l t o i n v e r t

n o r m a l l y i n one o r oo th s e g m e n ts o f t h e genome.

R e s t r i c t i o n e n d o n u c l e a s e maps were a l r e a d y a v a i l a b l e

o f HSV-1 HNA f o r A bal , i l i n d l l l , He oh I , Bgl 11 and H p a l , a n a o f

HSV-2 HHA f o r t n c s e enzymes and Kpnl ( W i l k i e , 1 9 76 ; S h a re

and Summers, 197'/; hors .e e t a l . , 1977 ; C o r t i n i and W i l k i e ,

1 9 7 8 ) . wap3 o f HSV-1 HHA f o r Kpnl , BamHI, n h o l and P v u I I ,

an a o f HSV-2 HHA f o r BamHI, were d e t e r m i n e d u s i n g t h rt

t e c h n i q u e s o f s i m u l t a n e o u s d i g e s t i o n w i t h two e n d o n u c l e a s e s ,

r e c l e a v a g e o f i s o l a t e d r e s t r i c t i o n f r a g m e n t s , and b l o t

h y b r i d i s a t i o n . P r e v i o u s l y r e p o r t e d s i z e h e t e r o g e n e i t y a t

t h e L t e r m i n u s and L-S j o i n t o f t h e two genomes was c o n f i r m e d .

The r e s u l t s s u g g e s t e d t h a t nSV-2 HHA i s 1 -2 x 1 0 ° l a r g e r i n

m o l e c u l a r w e i g h t i n t h e S’ seg m e n t t h a n HSV-1 HHA.

HSV-1 an a nSV-2 hha s n a r e a p p r o x i m a t e l y 50^ hom ology ,

ana PhV HHA p o s s e s s e s n o t g r e a t e r t h a n 8f* homology w i t h4

iiSV-1 HHA ( K i e f f e t a l . , 1972 ; B ronson e t a l . , 1972 ; Rand32ana B e n - P o r a t , I 9 6 0 ) , u y o r i d i s a t i o n o f P - l a b e l l e d

r e c o m b i n a n t p l a s m i d s c o n t a i n i n g HSV-1 o r HSV-2 DNA i n s e r t s t o

b l o t s t r i p s o f r e s t r i c t i o n e n d o n u c l e a s e d i g e s t s o f HSV-1 o r

iiSV-2 Hi.A snowed tuali t n e two genomes a r e c o l i n e a r , w i t h i n

ti ie r e s o l u t i o n a t t a i n e d i n t h e s e e x p e r i m e n t s . H y b r i d i s a t i o n

o f ^‘‘P - l a b e l i ou !3V-1 o r nSV-2 HHA wo s i m i l a r b l o t s t r i p s

a l i o we u s e v e n r e ;io*is 01 t h e genome to be i d e n t i f i e d w h ich

a r c more hom ologous t n a n n e i , . n b o u r in g r e g i o n s . HHV—1 and

PuV jJLA a l s o showea j r j - j o r i iomoiogj bo t h e s e r e g i o n s o f

t h e nSV genome t n a n to o 'boo rs , b u t no s i g n i f i c a n t h o r o l o g y

b e tw e e n * S V i>iJA an a riOnV ±fuA was u w b e c t e u . Homologous r e g i o n s

p r o b a b l y r e f l e c t p.*eater c o n s e r v a t i o n o f t h e s t r u c t u r e s o f

p o l y p e p t i d e s encoaod by tnem . f i v e good c a n d i d a t e s f o r

c o n s e r v e d uSV-1 p o l y p e p t i a e s a r e t h e m a j o r HHA-binning; p r o t e i n ,

t h e m a j o r c a p s i u p r o t e i n , t h e DUA p o l y m e r a s e , and two

i m m e d i a t e - e a r l y p o l y p e p t i d e s , Vmw i h 175 and V|i;v/ I n 1 3 6 ' ( 1 4 3 ) .

f i y b r i d i s a t i o n o f c l o n e d nSV DU A f r a g m e n t s to b l o t s t r i p s o f

nnV-1 o r PhV r e s t r i c t i o n e n d o n u c l e a s e d i g e s t s shovvea t h a t

hom ologous r e g i o n s i n t h e L segm ent o f t h e HHV-1 genome a r e

o o l i n e a r w i t n t h e n s eg m en t o f t n e HSV-1 genome i n biie

a r r a n g e m e n t . Homologous r e g i o n s be tw een HSV an a PuV jjuA

were shown n o t to oe a r r a n g e d i n a s i m p l e c o l i n e a r f a s h i o n .

The ^cnome s t r u c t u r e s were a n a l y s e d o f more ' than a

h u n d r e d L S V -l /aS V -2 i n t e r t y p i c r e c o m b i n a n t s p ro d u c e d by

m a r k e r r e s c u e o f HSV-1 r a h w i t h HSV-2 r e s t r i c t i o n f r a g m e n t s

s p a n n i n g t h e L-o j o i n t . The r e c o m b i n a n t s p o s s e s s e u c r o s s o v e r s

p r e f e r e n t i a l l y i n Homologous regio**s. At l e a s t two o f t h e

genome a r r a n g e m e n t s (P and I g ) r e c o m b i n e d , a r e s u l t w h ich

d i s p r o v e s t h e e a r l i e r p r o p o s i t i o n t h a t o n l y one o f t h e s e

two a r r a n g e m e n t s i s a b l e to t a k e p a r t i n t h e g e n e r a t i o n o f

v i a b l e r e c o m b i n a n t s •

HSV hi.A i s b e r m i n a i l y r e d u n d a n t , p o s s e s s i n g a d i r e c t l y

r e p e a t e d s e q u e n c e o f 250 -300 b a s e p a i r s ( t h e a s e q u e n c e ) a t

t h e t e r m i n i which i s a l s o p r e s e n o i n i n v e r s e o r i e n t a t i o n a t

t h e L—b j o i n t ( S u e l d r i c k and j e r t h e l o t , 1 9 7 4 ; G ra f s ^ ro m e t a l . ,

1 j 74 a n a 1J7 3; , /aguer and Summers, 1 9 7 b ) . P i n e s t r u c t u r e

r e s t r i c t i o n enaon e l e a s e «ups o f t h e L-S j o i n t r e g i o n o f HSV-1

m u iSV-2 HHA v.er d c t e r m i n u a , an a n u c l e o t i d e s e q u e n c e s were

a o r i v e a u s l u c ivn .sa i i-rn,u >.cn b s • H e l a b i v e bo b n j s e

s e q u e n c e s ' tne mor.ani a r e i n b o t h c a s e s l o c a l e d c l o s e

t o a s n o r t diroc i repeat o. 1 7 -2 1 Ouse j j a . t r s a t tne o -a and

a - c j u n c t i o n s . Tne . .oV'-l n - o j o i n t r e g i o n c o n t a i n . ; irhrce

s e p a r a t e t u n u e dircc t r e i t e r a t i o n s o i s n o r t s e q u e n c e s

( 1 2 , 16 and 17 e a s e p a i r s ) , and t n e s i z e h e t e r o g e n e i t y o i

v i r i o n LLA i n t n e a a . n c s e q u e n c e s i s a l m o s t c e r t a i n l y

due to v a r i a b l e copy n um bers o f t h e s e r e p e a t e d u n i t s .

A s e c o n d t y p e o f s i x e h e t e r o g e n e i t y was f o u n d t o be due to

i n s e r t i o n o f an a d d i t i o n a l a s e q u e n c e i m m e d i a t e l y a d j a c e n t t o

t h e a s e q u e n c e a t t h e L-S j o i n t . The r e g i o n c o d i n g f o r t n e

3> t e r m i n u s o f HSV-1 VuW I d 175 mAhA was i d e n t i f i e d

a p p r o x i m a t e l y SOU b a s e p a i r s f ro m t h e a s e q u e n c e , b u t i t i s

l i h e l y t h a t t n e a s e q u e n c e and i m m e d i a t e l y a d j a c e n t r e g i o n s

do n o t code f o r p o l y p e p t i d e .

The iISV-i /i iSV-2 i n t e r t y p i c r e c o m b i n a n t B x l ( 2 8 - 1 ) p o s s e s s e s

a m a j o r i t y o f v i r i o n i)l\IA m o l e c u l e s w i t h t h e L s eg m en t i n one

o r i e n t a t i o n ( . f ix ed i n h ) , w h e r e a s t n e S segm ent i n v e r t s

n o r m a l l y ( P r e s t o n e t a l . , 1 9 7 8 ) . An a n a l y s i s o f B x l ( 2 8 - 1 )

and tw e n ty s u b c l o n e s t h e r e o f , some o f w h ic h a r e f i x e d and some

o f w hich i n v e r t n o r m a l l y i n L, showed t h a t f i x e d genomes a r e

t o t a l l y h e t e r o l o g o u s f o r fib^ and 1 t h , and t h a t genoir.es w h ich

i n v e r t n o r m a l l y p o s s e s s a d d i t i o n a l c r o s s o v e r s w h ich g e n e r a t e

r e g i o n s o f t y p e - s p e c i f i c homology b e tw een TR^ and IRg .

Homology be tw een a s e q u e n c e s i s s u f f i c i e n t t o a l l o w n o rm a l

i n v e r s i o n o f L, s u g g e s t i n g t h a t s e g m e n t i n v e r s i o n o c c u r s by a

s i t e - s p e c i f i c e v e n t i n o r n e a r t h e a s e q u e n c e . A l e s s

e x p e n s i v e a n a l y s i s u f n_>i, a r e c o m b i n a n t w hich i s f i x e d i n

L arid S, s u g g e s t s w a t a s i m i l a r r e q u i r e m e n t a p p l i e s t o t h e

3 s e g m e n t . As p r e d i c t e d , f i x e d b ; : l ( 2 8 - l ) s u b c l o n e s were a b l e

to g i v e r i s e l,o n o r m a l l y i n v e r t i n g p r o g e n y , but t h e r e v e r s e

c o n v e r s i o n was a s u o o s o r v e d . The a n a l y s i s was e x t e n d e d to

i n c l u d e a number oT o t n o r r e c o m b i n a n t s which were i h - i t i a l l y

t h o u g h t t o be h e t e r o l o g o u s i o r l i t J IK o r TRg/IRg, an a y e t

i n v e r t e d n o r m a l l y , n a c n o f t n e s e was fo u n d t o p o s s e s s

t y p e - s p e c i f i c homology b e tw een r e p e a t r e g i o n s by v i r t u e o f

p r e v i o u s l y u n d e t e c t e d c r o s s o v e r s . I n v e s t i g a t i o n om t n e

im m e d i a t e e a r l y p o l y p e p t i d e s i n d u c e d by 1 x 1 ( 2 8 - 1 ) s u b c l o n e s ,

i n c l u d i n g two w hich lac ' . : p a r t o f IKg o r TRp, showed t h a t

e x p r e s s i o n o f o n l y one o f t h e two r e p e a t s i s n e c e s s a r y f o r

g r o w th o f HSV i n v i t r o .

ABBiimV lATIOiMS

A u b r e v ia t io n s f o r m edia and s o lu t i o n s a r e g iv e n in th e

M a te r ia ls and th o se f o r th e genome r e g io n s o f HSV are

g iv e n on page 7 and in P ig u re A l . l . A l l tem p era tu res a re

g iv e n in d e g r e e s c e n t ig r a d e .

A a d e n in e -c o n ta in in g m o ie ty

ACV a c y c lo v ir

ATP a d e n o s in e tr ip h o s p h a te

111K baby ham ster k id n ey c e l l s

H..V b o v in e m a m m ilit is v ir u s

bp b ase p a ir s

C c y t o s in e - c o n t a in in g m o ie ty

00V ch a n n e l c a t f i s h v ir u s

cPhA com plem entary DNA

CiviV (human) c y to m e g a lo v ir u s

c . p . e . c y to p a th ic e f f e c t

c*p .m . c o u n ts p er m inute

dATP d eo x y a d en o sin e t r ip h o s p h a te

dCTP d e o x y c y t id in e tr ip h o s p h a te

dGTP d co x y g u a n o sin e t r ip h o s p h a te

PMSO d im e th y l .su lp h o x id e

Phase d e o x y r ib o n u c le a se

dPyK d eo x y p y r im id in e k in a se

dTTP th ym id in e t r ip h o s p h a te

LBV L p s te in -P a r r v ir u s

KDTA e th y ie n e d ia m in e t e t r a - a c e t i c a c id

BHY- 1 oquid h e r p e s v ir u s 1

LllV-2 oquid h e r p e s v ir u s 2

G g u a n in e -c o n ta in in g m o ie ty

GC c o n t e n t a o l e s p e r c e n t deoxyguano s in e p lu su eoxycy t i d i n e m o i e t i e s

HCkiV human c y to m e g a lo v ir u s

hr hours

HSV n erp es s im p lex v ir u s

HSV-1 n erp es s im p le x v ir u s ty p e 1

HSV-2 h erp es s im p lex v ir u s ty p e 2

HVA n e r p e s v ir u s a t e l e s

HVP h e r p e s v ir u s p a p io

HVS h e r p e s v ir u s s a im ir i

HVT h e r p e s v ir u s o f tu r k e y s

IK im m ediate e a r ly

kb k i lo b a s e s

kbp k i lo b a s e p a ir s

LHV Lucke h e r p e s v ir u s

MCMV m urine c y to m e g a lo v ir u s

MCP m ajor c a p s id p r o t e in

MJ)J3P m ajor H NA-binding p r o t e in

MTV h a r e k ’ s d i s e a s e v ir u s

m in m in u tes

m .o . i . m u l t i p l i c i t y o f i n f e c t i o n

mKNA m essen ger RHA

m .wt. m o le cu la r w e ig h t

n n u c le o t id e s

N u n s p e c if ie d n u c le o t id e (A, 0 , G or T)

H?40 h o n id e t P4-8

02 ounces

PAA p h o sp h o n o a ce tic a c id

p .f # u . p la q u e -fo r m in g u n i t s

p . i . p o s t i n f e c t i o n

PiV p se u d o r a b ie s v ir u s

Pa p u rin e

Py p y r im id in e

RNase r ib o n u c le a s e

r .p .m .

rRNA

SGMV

SI)S

SV40

syn

syn +

T

T■Lm

tRNA

t s

U

UV

v o l / v o l

v z v '

W't

w t / v o l

X

r e v o lu t io n s p er m inute

r ib o so m a l RHA

sim ia n c y to m e g a lo v ir u s

sodium d o d e c y l s u lp h a te

sim ia n v ir u s 40

s y n c y t ia l

n o n - s y n c y t ia l

th y m id in e -c o n ta in in g m o ie ty

m e lt in g tem p era tu re

t r a n s f e r RIM; A

t e m p e r a t u r e - s e n s i t iv e

u n i t s , or u r a c i l m o ie ty

u l t r a v i o l e t

volu m e/volu m e

v a r i c e l l a - z o s t e r v ir u s

w eig h t

w e ig h t/v o lu m e

u n s p e c i f ie d n u c le o t id e (A, C, G o r T)

Arrangem ent o f d a ta

T a b le s and th en F ig u r e s have been in c lu d e d a f t e r

each P a r t or S e c t io n , and a d d i t io n a l c o p ie s o f key F ig u r e s

have been in c lu d e d a t th e i n s id e back cover*

i' A j-i i ‘ ii

1 i. U! H O j j U C T I O 14

Knowledge o f h e r p e s v ir u s e s h as exx)anded c o n s id e r a b ly

d u r in g th e p a s t iew y e a r s , and th e r e fo r e th e f o l lo w in g x^&ges

s t r e s s h erx^ esvirus genome s tr u c tu r e and th e e x p r e s s io n o f th e

HSV genome in th e l y t i c a l l y in f e c t e d c e l l . N e c e s s a r i ly ,

o th e r a s i> e c ts o f eq u a l i n t e r e s t and im p o rta n ce a re d e a l t

w ith o n ly b r i e f l y .

THE HEHPESVIKUSES

C l a s s i f i c a t i o n and p a th o g e n ic i ty

h e r p e s v ir u s e s are c h a r a c t e r is e d by v ir io n m orpnology, •

genome s i z e and s i t e o f v ir u s r e p l i c a t i o n . P a r t i c l e s w ith

h e r p e s v ir u s m orphology have been o b serv ed in a w ide v a r ie t y

o f o rg a n ism s: fu n g i (Kazama and S c h o r n s te in , 1 9 7 2 j , f i s h

( e . g . ch a n n e l c a t f i s h v ir u s , CCV; Yi/olf and D a r lin g to n , 1971) >

am phib ia ( e . g . Lucke h e r p e s v ir u s , LEV; S ta c k p o le , 1 9 6 9 ) , b ir d s

( e . g . h a rem 's d i s e a s e v ir u s , MDV; C h u r c h il l and B ig g s , 1 9 6 7 ) ,

m a rsu p ia ls ( e . g . Parma w a lla b y v ir u s ; F in n ie e t a l . , 1 9 7 6 )

and a la r g e number o f mammals. S e v e r a l h e r p e s v ir u s e s have been

i s o l a t e d from non-human p r im a te s ( e . g . h e r p e s v ir u s s a im ir i ,

iiVE, and herx^esvirus a t e l e s , HVA; Barahona e t a l . , 1974;

h e r p e s v ir u s p a p io , HVP; Falk egt a l . , 1 9 7 6 ) , and f i v e from

humans (herxjes s im p lex v ir u s ty p e s 1 and 2 , HSV-1 and HSV-2;

human c y to m e g a lo v ir u s , ilGr.iV; E p s te in -B a r r v i r u s , EBV;

v a r i c e l l a - z o s t e r v ir u s , VEV).

The number o i members o f th e fa m ily H e r p e s v ir id a e h a s

in c r e a s e d aiJuce d u rin g r e c e n t y e a r s and w ith i t th e la b o u r

in a s s ig n in g an in t e r n a t io n a l n o m en cla tu re (Hoizman e t a l . ,

1978; M athews, 1 9 7 9 ) . Three s u b f a m il ie s have been su g g e s te d

wnich a re d is t in g u is h e d by h o s t r a n g e , f e a t u r e s o f th e

r e p l ic a t - iv e c y c l e , and, to some e x t e n t , genome o r g a n is a t io n .

The a lp h a h e r p e s v ir in a e p o s s e s s a v a r ia b le h o s t ran ge and a

r e l a t i v e l y sh o r t r e p l i c a t i v e c y c l e , and in c lu d e iiSV -±, HSV-2,

eq u id herx>esv irus 1 (n h V - i; , su id n e r p e s v ir u s 1 or p se u d o r a b ie s

v ir u s (P hV ), aru oov id h e r p e s v ir u s 2 o r b ov in e mammi .JL t i s

v ir u s (BivjV ). A number o f o th e r c y to m e g a lo v ir u s e s a s w e l l a s

HCi/iV c o n s t i t u t e the b e ta h e r p e s v ir in a e . w hich have a narrow

h o s t ran ge and a r e l a t i v e l y lo n g r e p r o d u c t iv e c y c l e , and

u s u a l ly grow bes u in f i b r o b l a s t s . EBV, 1IVP, HVS and HVA are

cou n ted among th e g a m a a h e r p e sv ir in a e , w hich h ave a narrow

h o s t ran ge and r e p l i c a t e in ly m p h o b la s to id c e l l s . These

v ir u s e s a re s p e c i f i c f o r e i t h e r B - o r T -ly m p h o c y te s .

B i o l o g i c a l a s p e c t s o f th e human h e r p e s v ir u s e s have

been s tu d ie d in some d e t a i l . HSV-1 and HSV-2 a re r e la t e d

g e n e t i c a l l y and a n t i g e n i c a l l y , b u t form two d i s t i n c t s e r o ty p e s

(Plummer, 1964; P a u ls and D ow dle, 1 9 6 7 ) . HSV-1 i s r e s p o n s ib le

fo r v e s ic u l a r J c s io n s o f th e l i p s and mouth and f o r o c u la r

h e r p e t ic k e r a t i t i s . More s e v e r e symptoms, and a ls o

g e n e r a l is e d f a t a l i n f e c t i o n , have been d e s c r ib e d . HSV-1 i s

m a in ta in ed in a l a t e n t form in th e t r ig e m in a l , and som etim es

o th e r , sym x>athetic g a n g l ia (B a r in g er and Sw oveland , 1973;

L on sd a le e t a l . , 1 9 7 9 ) . The v ir u s may be r e a c t iv a t e d

p e r i o d i c a l l y le a d in g to r e c u r r e n t sym ptom s. The m echanism s

by w h ich la t e n c y i s e s t a b l i s h e d and m a in ta in e d , and by w hich

r e a c t iv a t io n and r e c u r r e n c e tak e p la c e , are n o t known.

V iru s la t e n c y i s a f e a tu r e o f s e v e r a l , i f n o t a l l , h e r p e s v ir u s e s

anu t h e i r p a th o g e n e s is .

iiSV-2 i s s e x u a l ly t r a n s m it te d , l e s i o n s o c c u r r in g i n

the g e n i t a l r e g io n . There i s an a s s o c ia t io n betw een HSV-2

ana c e r v i c a l carcinom a in women (N aib e t a l . , 1 9 6 6 ; Uawls

e t a l . , 1 9 6 c i). ru..iour t is s u e has been shown, in a t l e a s t

so lie c a s e s , to o p p ress JiSV-2 s p e c i f i c a n t ig e n s and to c o n ta in

RhA com plem entary oo HbV-2 l)HA (R oyston and A u r e lia n , 1970;

P r en k e l e t a l . , 1972; E g lin e t a l . , 1 9 8 1 ) . Both HSV-1 and

HSV-2 c a u se m o rp h o lo g ic a l tr a n sfo r m a tio n o f ro d en t c e l l s in

v i t r o , e i t h e r a s u e b i l ib a te d v ir u s or a s HHA fra g m en ts

(B u ff and Rapp, 1971; D arai and Munk, 1973;

MaeNab, 1 9 7 4 ; Camacho and S p ea r , 1 978;

R eyes e t a l . , 1979; J a r iw a lla e t a l . , 1 9 80; G allow ay and

M cD ougall, 1981; I . Cameron, p e r so n a l co m m u n ica tio n ).

Tumours can be in d u ced in e x p e r im e n ta l a n im a ls by in o c u la t io n

w ith in v i t r o tran sform ed c e l l s , b u t n o t by i n f e c t i o n w ith

v ir u s p a r t i c l e s . The HSV-1 tr a n sfo r m in g r e g io n has been

mapped a t 0 .3 1 - 0 .4 2 f r a c t i o n a l genome u n i t s , and t h a t o f HSV-2

a t e i t h e r 0 . 43- 0 .3 8 or 0 . 58- 0 . 6 2 , d ep en d in g on th e tr a n sfo r m a tio n

a s s a y em p loyed . The n a tu r e o f tr a n s fo r m a tio n by HSV a t th e

m o le c u la r l e v e l rem ain s o b sc u r e .

1ICMV i n f e c t i o n i s u s u a l ly m ild , a lth o u g h i t may be

s e v e r e in n e o n a te s , and t h i3 v ir u s h a s been a s s o c ia t e d w ith

K a p o s i’ s sarcom a (G ira ld o e t a l . , 1 9 7 5 ) . VZV c a u s e s c h ick e n

p o x , u s u a l ly in c h ild h o o d , and, in a d u lth o o d , a p a in f u l l o c a l

v e s ic u l a r c o n d it io n known a s s h i n g l e s . VZV h a s been r e p o r te d

to tra n sfo rm c e l l s in v i t r o (G elb e t a l . , 1 9 8 0 ) . EBV i s

B -ly m p h o cy te s p e c i f i c and c a u s e s i n f e c t i o u s m o n o n u c le o s is

and i s in v o lv e d in th e a e t io lo g y o f two tum ours: B u r k i t t f s

lymphoma, p erh ap s prom oted by endem ic m a la r ia , and

n a so p h a ry n g e a l ca rc in o m a , in a s s o c i a t io n w ith a g e n e t ic

p r e d is p o s i t io n (Old e t a l . , 1966; H enle e t a l . , 1968 and 1 9 6 9 ) .

PRV i s a p a r a s i t e o f sw ine and i s u s u a l ly in a p p a r e n t ,

but g iv e s r i s e to th e a la rm in g symptoms o f ,fmad i t c h ” and

then d e a th in c a t t l e . EHV-1 c a u s e s p reg n a n t m ares to a b o r t .

Those two v i r u s e s have n o t y e t been im p lic a te d in

tu m o u r tg e n e s is in v iv o , in c o n t r a s t w ith s e v e r a l o th e r n o n -

human h e r p e s v ir u s e s . i.DV c a u se s a h ig h ly c o n ta g io u s

n eu ro lym p h om atosis in c i i ic x e n s , now c o n t r o l le d by im m u n isa tion

w ith th e r e la t e d h e r p e s v ir u s o f tu r k e y s (HVT; Marek, 190'7;

P u rch ase e t a l . , 1 9 7 1 ) . LHV p rod u ces a r e n a l adenocarcinom a

in f r o g s , th e tumour and v ir u s grow th d e m o n str a tin g opposed

s e a s o n a l d ep en d en ce (L ucke, 1934; P a w c e tt , 1 9 5 6 ) . H erp esv ir u s

s i l v i l a g u s c a u s e s a ly m p h o p r o l i f e r a t iv e d i s e a s e in c o t to n

t a i l r a b b i t s (H in z e , 1 9 7 1 ) . The T -lym phocyte s p e c i f i c Hew

World s im ia n h e r p e s v ir u s e s , HVS and HVA, in v a r ia b ly produce

m a lig n a n t lymphomas in c e r t a in h e te r o lo g o u s p r im a te h o s t s

(M elendez e t a l . , 1969 and 1 9 7 2 ) .

S tr u c tu r e o f th e v ir io n

The h e r p e s v ir io n co m p r ise s fo u r m o r p h o lo g ic a lly d i s t i n c t

s t r u c t u r e s : th e c o r e , c a p s id , tegum ent and e n v e lo p e . The co r e

c o n t a in s th e d o u b le -s tr a n d e d DNA genome (E p s te in , 1962a;

B en -P o ra t and K aplan, 1962) t o r o i d a l l y arran ged around a

c e n t r a l p r o te in a c e o u s m a tr ix (C h a i, 1971; F u rlo n g e t a l . >

1972; H a z e r ia n , 1974; H eine and C o t t le r -F o x , 1 9 7 5 ) .

P leom orp h ic fo rm s, p erh ap s r e p r e s e n t in g d i f f e r e n t d ev e lo p m e n ta l

s t a g e s , h ave a l s o been o b se rv ed ( N i i and Y asuda, 1975; Okada

e t a l* , 1 9 7 9 ) . The ic o s a h e d r a l c a p s id w h ich surrou n d s th e

co r e i s a p p r o x im a te ly 100 nm in d ia m e te r , and c o n s i s t s o f

162 cap so m eres i n 5 :3 :2 a k ia l symmetry (W ildy e t a l . , I 9 6 0 ) .

The ca p so m eres a re h o llo w e lo n g a te d p r ism s , and in te r c a p s o m e r ic

f i b r i l s h ave been o b serv ed ( W ildy e t a l . , 1 960; Vernon e t a l . ,

1 9 7 4 ) . A f ib r o u s la y e r known a s th e tegum ent su rrou n d s th e

c a p s id (Hoizman and F u r lo n g , 1 9 7 4 ) , th e w id th o f w hich i s

d eterm in ed a t l e a s t- in p a r t by th e v ir u s (McCombs e t a l . , 1 9 7 1 ) .

The f r a g i l e v ir io n en v e lo p e c o n s i s t s o f a t r i la m e l l a r

membrane w ith s p ik e s p r o j e c t in g from th e o u te r s u r fa c e

( Wildy e t a i . , 19 oO)•

H e r p e s v ir io n s c o n ta in 1 8 -3 3 p o ly p e p t id e s o f m .w t.

1 1 ,0 0 0 -2 9 0 ,0 0 0 (S p ear and Roizm an, 1972; H eine e t a l . , 1974;

Perdue e t a l • , 1974; S t e v e ly , 1975; S tm a d and A u r e lia n ,

1976; Kim e t a l . , 1976a and b; D o ly n iu k e t a l . , 1976; D ixon

and P a rb er , 1 9 8 0 ) . About h a l f o f th e s t r u c t u r a l p r o t e in s o f

HSV-1 have been a s s ig n e d r e l a t i v e p o s i t i o n s in th e v ir io n

on th e b a s i s o f ex p er im en ts in v o lv in g s e l e c t i v e rem oval o f

p o ly p e p t id e s by d e te r g e n t tr e a tm e n t (Roizman and F u rlo n g ,

1 9 7 4 ) , d i f f e r e n t i a l c h e m ic a l tr e a tm e n t (O lsh ev sk y and B eck er ,

1972; Roizman and F u r lo n g , 1974) and v i r u s n e u t r a l i s a t i o n

(P o w e ll e t a l . , 1974; Cohen e t a l . , 1 9 7 8 ) .

The e n v e lo p e c o n ta in s m ost o r a l l o f th e v ir u s -c o d e d

s t r u c t u r a l g ly c o p r o t e in s (S p ea r and Roizm an, 1 9 7 2 ) , c e l l

l i p i d s (A sher e t a l • , 1 9 6 9 ) , an ATPase p ro b a b ly o f c e l l u l a r

o r ig in (E p s te in and H o lt , 1 9 6 3 ) , and sp erm id in e (G ibson and

Roizm an, 1 9 7 1 ) . In c o n t r a s t to o th e r v i r i o n p r o t e in s ,

e l e c t r o p h o r e t ic p r o f i l e s o f c a p s id p r o t e in s from v a r io u s

h e r p e s v ir u s e s a r e s im i la r . Cohen e t a l . (1980b) i d e n t i f i e d

sev en m ajor c a p s id p o ly p e p t id e s in HSV-1, r a n g in g in m .w t.

from 1 2 ,0 0 0 to 1 5 5 ,0 0 0 , and Zweig e t a l . (1 9 7 9 ) showed t h a t

th e m ajor c a p s id p r o t e in ( 1 5 5 , 0 0 0 ) p a r t i c ip a t e s in d is u lp h id e

b on d in g in th e i n t e g r a l v i r i o n s t r u c t u r e . G ibson and

Roizman (1972 and 1974) p r e se n te d e v id e n c e f o r th e p r o c e s s in g

o f a p h o sp h o r y la te d c a p s id p r o te in to a l e s s t i g h t l y bound

form d u r in g v ir u s m a tu r a tio n . A c y c l i c n u c le o t id e -

in d ep e n d en t p r o t e in k in a s e i s ca p a b le o f p h o sp h o r y la t in g

ab ou t sev en s t r u c t u r a l p o ly p e p t id e s , in c lu d in g a c a p s id

p r o t e in (R u b en ste in e t aJL., 1972; le m a s te r and Roizm an, 1 9 8 0 ) .

The s ig n i f i c a n c e in v ir u s m a tu ra tio n and i n f e c t i v i t y o f th e s e

o b s e r v a t io n s i s n o t known. P o ly p e p t id e s w hich have n o t been

lo c a t e d in th e c a p s id o r en v e lo p e a re th o u g h t to r e s id e in

th e tegu m en t. Only one p r o te in h a s been s u g g e s te d a s a

s t r u c t u r a l com ponent o f th e c o r e , in a d d it io n to th e p o ly a m in e ,

sperm in e (G ib son and Roizm an, 1 9 7 1 ) .

S tr u c tu r e o f th e HSV genome

DNA e x tr a c te d from HSV-1 v i r i o n s i s a l i n e a r d u p lex

w ith a m .w t. o f a p p ro x im a te ly 100 x 1 0 5 a s d eterm in ed by

s e d im e n ta t io n , e le c t r o n m ic r o s c o p ic , and r e s t r i c t i o n

e n d o n u c le a se a n a ly s e s (B eck er e t a l . , 1 968; K ie f f e t a l • ,

1971; W ilk ie , 1973; G rafstrom e t a l . , 1 9 7 4 ; Wadsworth e t a l . ,

1975; C lem en ts e t a l . , 1976; W ilk ie , 1 9 76; Skare and Summers,

1 9 7 7 ) . I t h a s a d eo x y g u a n o sin e p lu s d e o x y c y t id in e (GC) c o n te n t o f

67 ft ( K ie f f e t a l . , 1 9 7 1 ) and, u n l ik e c e l l DNA, d o e s n o t have

a s c a r c i t y o f th e n u c le o t id e d o u b le t CpG (Subak-Sharpe e t a l . ,

1 9 6 6 ) . B oth s in g l e and d o u b le stra n d ed DNA a re i n f e c t i o u s

in c e l l m o n o la y ers (Lando and R y h in er , 1 9 6 9 ; Graham e t a l . ,

1973; S h e ld r ic k e t a l . , 1973; W ilk ie e t a l . , 1974; P a rb er ,

1976; Stow and W ilk ie , 1 9 7 6 ) .

The l a b i l i t y o f HSV-1 DNA in a l k a l i h a s been a t t r ib u t e d

to r ib o n u c le o t id e s c o v a le n t ly bound t o th e DNA, d e te c te d by

th e in c o r p o r a t io n o f r a d io a c t iv e u r id in e i n t o m ature DNA

( I l ir s c h and Vonka, 1974; M u ller e t a l . , 1 9 7 9 a ) . T h is

c o n c lu s io n i s in su p p o rt o f th e work o f G ordin e t a l . (1 9 7 3 )

who r e p o r te d th a t formamide d en aturation ^ r e s u l t e d in i n t a c t

s in g l e s t r a n d s . On th e o th e r hand, Hyman e t a l . (1 9 7 7 )

found t h a t th e m a jo r ity o f a l k a l i - l a b i l e s i t e s c o u ld be

r e p a ir e d by DNA p o ly m era se and DNA l i g a s e , and E cker and

Hyman ( 1 9 8 1 ) "claim ed th a t s in g l e s tr a n d s w ere fragm en ted even

u s in g g e l e l e c t r o p h o r e s i s sy stem s w hich l e a v e r ib o s e m o ie t i e s

i n t a c t . T hese a u th o r s co n c lu d ed th a t m ost o r a l l a l k a l i -

s e n s i t i v e s i t e s a re due to n ic k s o r gaps in th e s in g l e

s tr a n d s . T h is c o n c lu s io n , how ever, dep en d s on th e assu m p tio n

th a t th e DNA was n o t damaged upon r e l e a s e from v i r i o n s .

P ren k e l and Roizman (1 9 7 2 a )co n c lu d ed from r e n a tu r a t io n

s t u d ie s t h a t IISV-1 DNA c o n ta in s u n iq u e ly s i t u a t e d n ic k s on

o n ly one s tr a n d o f th e d u p le x , r a th e r l i k e b a c te r io p h a g e

15 . W ilk ie (1 9 7 3 ) co n c lu d ed from s im i la r e x p er im e n ts t h a t

in t e r r u p t io n s a re lo c a t e d on both s tr a n d s w ith e q u a l d i s t r i b u t i o n ,

and m oreover showed by com parison w ith b a c te r io p h a g e T5 DNA

th a t th ey a re random ly s i t u a t e d on both s tr a n d s (W ilk ie e t

a l . . 1 9 7 4 ) . The work o f E cker and Hyman (1 9 8 1 ) , w ith th e

c a v e a t m en tion ed a b o v e , i s in su p p o rt o f t h i s v ie w .

B en -P o ra t e t a l . (1 9 7 9 ) r e p o r te d th a t in t e r r u p t io n s in PRV

DUA a r e lo c a t e d a t random in th e s in g le s t r a n d s .

HSV-1 DNA i s n o t c i r c u l a r l y perm uted (H ir sc h e t a l . ,

1 9 7 5 ) . R e n a tu r a tio n o f HSV-1 DNA w hich h a s been t r e a te d w ith

p r o c e s s iv e e x o n u c le a s e s , such a s 3 ‘- e x o n u c le a s e I I I or

lambda 5 1- e x o n u c le a s e , r e s u l t s in th e fo rm a tio n o f d ou b le

s tra n d ed c i r c l e s ( S h e ld r ic k and B e r t h e lo t , 1 9 7 4 ; G rafstrom

e t a l . , 1974 and 1975; Wadsworth e t a l . , 1976.; Hyman e t a l . ,

1976; K udler and Hyman, 1 9 7 9 ) . T h is i s in t e r p r e t e d a s th e

p r e se n c e o f a d i r e c t r e p e t i t i o n a t th e two genome te r m in i ,

th e s i z e o f w hich f o r v a r io u s s t r a i n s o f HSV-1 h a s been

e s t im a te d to be 4 0 0 -7 5 0 0 bp, a lth o u g h th e more r e l i a b l e

e s t im a t e s p la c e i t betw een 400 and 1600 bp .

Upon ex a m in a tio n o f a n n ea led i n t a c t s i n g l e s tr a n d s o£

HSV-1 DNA in th e e le c t r o n m ic r o sc o p e , S h e ld r ic k and B e r th e lo t

(1 9 7 4 ) found s t r u c t u r e s c o n s i s t e n t w ith th e f o l lo w in g m od el.

The genome c o n s i s t s o f two c o v a le n t ly jo in e d segm en ts

(L and S ) , each o f w hich co m p r ise s a u n iq u e r e g io n (Uj, and

Ucj) bounded by in v e r te d r e p e t i t i o n s (TRp, and IRj,, TRg and IR s)*

a s sum m arised in P ig u re A l . l . They s u g g e s te d t h a t th e two

segm en ts m ight in v e r t by in tr a m o le c u la r r e c o m b in a tio n ,

th e re b y g e n e r a t in g fo u r d i s t i n c t genome a rra n g em en ts .

A lth ough th e s e w orkers supposed th a t TR^/IR^ and

- 8 -

are i d e n t i c a l , f u r th e r e le c t r o n m ic r o s c o p ic , d e n a tu r a t io n ,

and r e s t r i c t i o n en d o n u c lea se a n a ly s e s showed th a t th e two

s e t s o f r e p e a t s a re d i f f e r e n t (Wadsworth e t a l • , 1975;

Hayward e t a l . , 1 9 7 5 b ;D e liu s and C lem en ts , 1976; C lem ents

e t a l . , 1976; ’W ilk ie and C o r t in i , 1 9 7 6 ) . T hese r e s u l t s a ls o

con firm ed t h a t v ir io n DNA c o n ta in s fo u r eq u im o la r genome

arran gem en ts r e s u l t i n g from th e a b i l i t y o f L and S to in v e r t

r e l a t i v e to each o t h e r . The e s ta b lis h m e n t o f r e s t r i c t i o n

en d o n u c lea se maps showed t h a t h a l f - and q u a r te r -m o la r

fra g m en ts map a t th e te r m in i o f L and S and t h e i r m utual

j o in t , a s p r e d ic t e d by th e model and i l l u s t r a t e d in F ig u re ■

A i . l (W ilk ie , 1 976; W ilk ie e t a l . , 1 9 7 7 a ;S k a re and Summers,

1 9 7 7 ) .

The te r m in a l r e p e t i t i o n o f HSV-1 DNA, o r a se q u e n c e ,

i s a l s o p r e s e n t in in v e r s e o r ie n t a t io n a t th e j o i n t betw een

L and S ( f ig u r e A l . l ) . Wagner and Summers (1 9 7 8 ) o b ta in e d

d e t a i l e d r e s t r i c t i o n maps o f t h i s r e g io n , and e s t im a te d th e

s i z e o f th e a seq u en ce in HSV-1 s t r a in KOS to be 265 bp .

Two ty p e s o f s i z e h e t e r o g e n e i t y a re a s s o c ia t e d w ith th e

te r m in i and j o in t r e g io n . The f i r s t c o r r e sp o n d s to i n s e r t i o n s

o f a p p r o x im a te ly 300 bp, th o u g h t to c o n ta in th e a se q u e n c e ,

a t th e L term in u s and j o in t (W ilk ie , 1976; Wagner and Summers,

1978; L ocker and F r e n k e l, 1 9 7 9 b ) . T h is h a s a l s o been

o b serv ed f o r th e HSV-2 genome (W ilk ie e t g l« » 1 9 7 7 a ) . The

secon d ty p e o f h e te r o g e n e ity c o n s i s t s o f v a r ia b le i n s e r t i o n s

o f 1 0 -5 0 bp w ith in th e a seq u en ce and th e a d ja c e n t c

(TK^/IKg) seq u en ce ( ’Wagner and Summers, 1 9 7 8 ) .

R en a tu r a tio n o f e x o n u c le a s e - t r e a t e d HSV—1 DNA r e s u l t s ,

in some in s t a n c e s , in a sm a ll lo o p a t one te r m in u s , in t e r p r e te d

a s th e p r e se n c e o f p a r t o f th e w hole o f th e a seq u en ce in

in v e r s e o r ie n t a t io n a few hundred bp away from th e term in u s

(W adsworth‘e t a i . , 1976; Hyman e t a l . , 1 9 7 6 ) . W ilk ie and

C o r t in i (1 9 7 6 ) s u g g e s te d from experim ents- in v o lv in g en d -

l a b e l l i n g o f IiSV-1 DNA th a t t h i s s t r u c t u r e i s p r e s e n t a t

th e L te r m in u s . H i l l e r e t a l . (1 9 8 0 ) have o b se rv ed a w ide

v a r ie t y o f fo ld b a c k and s te m -lo o p s t r u c t u r e s in r e a n n e a le d

s in g l e s tr a n d s , some o f w hich co rresp o n d to r e c o g n is e d

r e p e a t s w ith in th e genom e, and o th e r s o f w h ich a re o f unknown

s i g n i f i c a n c e .

K udler and Hyman (1 9 7 9 ) o b serv ed t h a t p a r t i a l l y

d e p r o t e in is e d DNA i s more r e s i s t a n t to 3 1- e x o n u c le a s e than

to 5 *- e x o n u c le a s e , and su g g e s te d t h a t t h i s was due to a

p r o t e in bound a t th e 3 1 t e r m in i . In d ee d , fo u r s p e c i f i c

p r o t e in s rem ain n o n -c o v a le n t ly a s s o c ia t e d w ith HSV-l DNA

f o l lo w in g d e te r g e n t tr ea tm e n t (Hyman, 1 9 8 0 ) . Wu e t a l* (1 9 7 9 )

a t ta c h e d d in itr o p h e n y l (DNP) grou p s to th e bound p r o t e in s

and v i s u a l i s e d them in th e e le c t r o n m icro sco p e by tr e a tm e n t

w ith an anti-D N P im m u n og lob u lin . They found p r e f e r e n t i a l

b in d in g c l o s e to th e two te r m in i and a t two p o s i t i o n s

c o r r e sp o n d in g to th e a seq u en ce a t th e j o i n t , and p rop osed

th a t a p r o t e in s p e c i f i c a l l y i n t e r a c t s w ith th e a s e q u e n c e .

The s ig n i f i c a n c e o f t h i s o b s e r v a t io n h a s y e t to be e lu c id a te d *

HSV-2 DHA h as a 2 ft h ig h e r GC c o n te n t th an HSV-l DNA

(G oodheart e t a l . , 1968; K ie f f e t a l . , 1971; H a ll ib u r to n ,

1 9 7 2 ) . A p p rox im ately 50 o f HSV-l and HSV-2 DNA se q u e n c e s

a re hom ologous w ith 6 5 -8 5 ^ m atch in g o f n u c le o t id e s ( K ie f f

e t a l . , 1972; F ren k e l e t a l . , 1973; Sugino and K in gsb u ry , 1 9 7 6 ) .

The genom es o f th e two s e r o ty p e s a re s t r u c t u r a l l y v e r y

s im i la r , but p o s s e s s d i s t i n c t i v e arran gem en ts o f r e s t r i c t i o n

s i t e s (Hayward e t a l • , 1975a and b; Skare e t* a l * , 1 9 7 5 ;

C o r t in i and W ilk ie , 1 9 7 8 ) . These d i f f e r e n c e s have been

e x p lo i t e d a s a d ia g n o s t ic t o o l f o r i d e n t i f i c a t i o n o f HSV-l

and HSV-2 i s o l a t e s and f o r s tu d y in g e p id e m io lo g ic a l a s p e c t s

o f o v e r t and l a t e n t i n f e c t i o n s in man (linnem an e t a l . , 1978;

Buchman e t a l . , 1978 and 1980; L o n sd a le e t a l • , 1979 and 1980;

L o n sd a le , 1 9 7 9 ) . R e s t r ic t io n fra g m en ts r e p r e s e n t in g th e

m a jo r ity o r e n t i r e t y o f th e IISV-1 and HSV-2 genom es have

r e c e n t ly been c lo n e d in t o b a c t e r ia l p la s m id s , b o th in Glasgow

and e lse w h e r e (P o s t e t a l . , 1980; G allow ay and Sw ain , 1980;

G old in . e t a l . , 1 9 8 1 ) .

S tr u c tu r e s o f o th e r h e r p e s v ir u s genom es

A lth ough a number o f genome s t r u c t u r e s have been

d em o n stra ted among th e h e r p e s v ir u s e s , in each c a s e a l i n e a r

d u p le x , t h e s e may be r e p r e s e n te d i n c i r c u l a r form in o rd e r to

show th e fo u r b a s ic o r g a n is a t io n s found among th o s e so f a r

a n a ly se d (F ig u r e A 1 .2 ) . GC c o n te n ts o f h e r p e s v ir u s DNAs are

g iv e n in F ig u re A 1 .3 .

Two subgroups are. in c lu d e d in group I b e c a u s e , a lth o u g h

th ey a re d i s t i n c t , i t i s p o s s ib l e t h a t th e y r e f l e c t a b a s ic

s i m i l a r i t y . The f i r s t i s common to H SV -l, HSV-2, BMV and

UCMV. BkV DNA p o s s e s s e s 1 4 # hom ology w ith HSV-l DNA (S te r z

e t a l . , 1 9 7 4 ) and has a ra.wt. o f a p p ro x im a te ly 90 x 1 0 ^,

p o s s e s s in g s m a lle r in v e r te d r e p e a t s th an HSV-l DNA (Buchman

and Roizm an, 1 9 7 8 a ) . The BIvlV genome h as a te r m in a l r e p e t i t i o n

o f a p p r o x im a te ly 1000 bp (Buchman and Roizm an, 1 9 7 8 b )• The

genome o f HCiuV has a m.wt*. o f 150 x 10^ (D em archi e t a l . , 1978

G eelen e t a l . , .1978; S t in s k i e t a l . , 1979; W e s ts tr a te e t a l . ,

1 9 8 0 ) , a lth o u g h i t was e a r l i e r r e p o r te d t h a t HCMV DNA h a s a

m .w t. o f 100 x 10^ (S arov and Friedm ann, 1 976; K i lp a tr ic k

e t a l . , 1 9 7 6 ; Huang e t a l . , 1 9 7 3 ) . K i lp a tr ic k and Huang

(1 9 7 7 ) d e t e c te d both s i z e c l a s s e s , and Lakeman and Osborn

(1 9 7 9 ) a fu r th e r c l a s s o f 130 x 1 0 ^. i t i s c l e a r th a t th e

s i z e c l a s s o f 150 x 1 0 6 r e p r e s e n ts th e f u l l l e n g th genome,

b oth from d e te r m in a t io n s o f i t s i n f e c t i v i t y and from

r e s t r i c t i o n maps (G eelen e t a l . , 1978; Lakeman and O sborn,

1979; W e s ts tr a te e t a l . , 1 9 8 0 ) . The s m a lle r s i z e s o f DNA

- 11 -

m o le c u le s a re thought to r e s u l t from d e f e c t i v e v i r i o n s .

The HCMV genome i s s t r u c t u r a l ly s im i la r to th a t o f HSV-l

(W e s ts tr a te e t a l . , 1 9 8 0 ) . and Us a r e la r g e r than in H SV -l,

and th e r e g io n o f l e a s t hom ology betw een two c l o s e l y r e la t e d

s t r a i n s i s in rfR g/IR g (com p arison o f th e r e s u l t s o f P r i t c h e t t ,

1980 w ith th e r e s t r i c t i o n maps o f W e s ts tr a te e t a l . , 1 9 8 0 ) .

Jean e t a l . (1 9 7 8 ) have p o s tu la te d from i n d i r e c t e v id e n c e t h a t

HCMV PH A i s t e r m in a l ly red u n d a n t. R e s t r i c t io n p r o f i l e s o f

s im ia n CMV (SCMV) i s o l a t e s d i f f e r s i g n i f i c a n t l y from th o se

o f HCMV ( iC ilp a tr ic k e t a l . , 1 9 7 6 ) , and th e r e h as been a t

l e a s t one c a s e o f a c y to m e g a lo v ir u s i s o l a t e d from a human

p a t ie n t who d ied from c l i n i c a l e n ce p h a lo p a th y w h ich bore

a lm o st t o t a l n u c le ic a c id hom ology w ith SCMV s t r a i n s but none

w ith HCMV (Huang e t a l . , 1 9 7 8 ) . Mosmann and Hudson (1973)

r e p o r te d th a t th e DNA o f m urine CMV (MCMV) h a s a m .w t. o f6132 x 10 . T aking in t o a cc o u n t t h e i r u n d e r e s t im a te o f th e

s i z e o f th e HSV-l genom e, t h i s i s e q u iv a le n t t o 150 x 10 .

However, genome s t r u c t u r e s o f MCMV, SCMV and th e c y to m eg a lo

v ir u s iilIV-2 ( Wharton e t a l * , 1981) have n o t been p u b lis h e d .

N e v e r t h e le s s , i t i s c l e a r t h a t th e HSV ty p e o f genome

o r g a n is a t io n i s found in r e p r e s e n t a t iv e s o f b o th a lp h a -

and b e t a - n e r p e s v ir in a e .

The secon d subgroup o f group I c o n t a in s th e PRV and

BIIV-1 genom es, w hich a re s im i la r in s i z e to t h a t o f HSV-l

but la c k in v e r te d r e p e a t s f la n k in g th e 1 seg m en t, and in

w hich o n ly S in v e r t s ( S t e v e ly , 1977; P o w e ll , 1979; B en -P orat

e t a l . , 1979; Soehner e t a l . , 1965; W halley e t a l . , in p r e s s ) .

There i s 110 d i r e c t e v id e n c e t h a t e i t h e r PRV o r EHV-1 DNA i s

t e r m in a l ly red u n d a n t, a s i t h as n o t proved p o s s ib l e to

c i r c u l a r i s e e i t h e r a f t e r e x o n u c le a se I I I tr e a tm e n t (Ben—P o r a t

e t a l . , 1979; P . S h e ld r ic k , p e r s o n a l co m m u n ica tio n ).

Group I I c o n ta in s EBV and i t s s im ia n c o u n te r p a r t , HVP.

The EBV genome has a m .w t. o f a p p r o x im a te ly 110 x lO *’3

( P r i t c h e t t e t a l . , 1975; Given and K ie f f , 1978 and 1 9 7 9 ) .

The te r m in i a re redundant and o f v a r ia b le s i z e , c o n ta in in g

up to 12 d i r e c t r e p e a t s o f a 500 bp seq u en ce (G iven and K ie f f ,

1 978; G iven e t a l . , 1979; Kimtner and Sugden, 1 9 7 9 ) , and , a s

w ith H SV -l, th e l i n e a r genome can be c i r c u l a r i s e d v i t r o by

a n n e a lin g f o l lo w in g e x o n u c le a se tr e a tm e n t (K in tn e r and Sugden,

1 9 7 9 ) . C ir c u la r DBA produced by r e p r e s e n t a t iv e s o f group I

has so f a r been d e te c te d o n ly in t h i s f a s h io n or in r e p l i c a t in g

IMA d u r in g l y t i c i n f e c t i o n , b ut E B V -transform ed c e l l s have

been shown to c o n ta in EBV genom es a s e p iso m a l c o v a le n t ly

c lo s e d c i r c l e s (L in d a h l e t a l • , 1 9 7 6 ) . The two u n iq u e

segm en ts o f th e l i n e a r genome do n o t in v e r t (G iven and K ie f f ,

1979; Hayward eti a l . , 1980) and a re se p a r a te d by a p p ro x im a te ly

10 d i r e c t r e p e a t s o f 3 kbp (IK ), w hich a r e G C -rich and n o t

hom ologous to th e te r m in a l r e p e a t s (Sugden , 1 977; Rymo and

Porsblum , 1978; Given and K ie f f , 1979; Hayward e t a l . , 1 9 8 0 ) .

G iven and K ie f f (1 9 7 9 ) d e te c te d in t e r n a l hom ology w ith in IR,

and co n c lu d ed th a t th e 3 kbp seq u en ce i t s e l f c o n ta in s a

r e p e a te d u n i t . They a l s o d e te c te d hom ology betw een IR and

two l o c a t io n s in L. The v a r io u s s t r a in s o f EBV d i f f e r in

t h a t some la c k se q u e n c es p o s s e s s e d by o th e r s ( P r i t c h e t t e t a l . ,

1975; Sugden e t a l . , 1 9 76; G iven and K ie f f , 1978; Rymo and

Porsblum , 1978; Raab-Traub e t a l , 1978 and 1980; Hayward e t

a l . , 1 9 8 0 ; Bomkamm e t a l . , 1980; H e l le r et. a l . , 1983a)..

Some s t r a i n s a re u n a b le to tra n sfo rm c e l l s , but i t rem a in s to

be shown p r e c i s e l y w h ich r e g io n s o f th e genome a r e e s s e n t i a l

fo r tr a n s fo r io a t io n .

HVP e x h i b i t s a p p ro x im a te ly 4 0 DNA hom ology w ith EBV,

a s do th e gam m aherpesvirinae o f o th e r Old World p r im a te s

such a s th e oran gu tan (h e r p e s v ir u s p o n g o ), th e ch im panzee

(h e r p e s v ir u s pan) and th e g o r i l l a (h e r p e s v ir u s g o r i l l a )

- 13 -

(G erber e t a l . , 1976; F a lk e t a l . , 1976; R abin e t g l . , 1978;

Heubauer e t a l . , 1979; Lee e t a l . , 1 9 8 0 ) . R e s t r i c t io n mapping

o f IIVP DNA and h y b r id is a t io n , u s in g in one c a s e c lo n e d EBV DNA

fr a g m e n ts , have shown t h a t th e two genom es a re s t r u c t u r a l ly

i d e n t i c a l and c o l in e a r (Lee e t a l . , 1981; H e l le r e t a l . , 1981b;

H e l le r and K ie f f , 1 9 8 1 ) .

The genom es o f HVS and HVA, w h ich a r e in group I H ,

b oth c o n s i s t o f a s in g l e u n iq u e r e g io n o f 3 5 # GC bounded on

b oth s id e s by m u lt ip le d i r e c t r e p e a t s o f a seq u en ce o f

a i> p rox im ately 1500 bp c o n ta in in g 7 0 $ GC ( F le c k e n s te in and

W olf, 1 9 7 4 ; F le c k e n s te in e t a l . , 1975 and 1978; BornXamm

e t a l . , 1 9 7 6 ) . As w ith EBV, H V S-transform ed c e l l s c o n ta in

e p iso m a l c lo s e d c i r c u la r HVS genom es, a lth o u g h such m o le c u le s

have a t l e a s t i n one c a s e been shown to co m p rise rea rra n g em en ts

o f th e HVS genome (Werner e t a l . , 1 9 7 7 ) . I a th e l i n e a r

genom e, th e te r m in a l r e p e a t s a re a p p r o x im a te ly c o n s ta n t in

num ber, a c c o u n t in g f o r 3 0 $ o f th e genome s i z e , but can be

d i s t r i b u t e d a sy m m e tr ic a lly a t th e two t e r m in i . The HVS and

HVA genom es have a m .w t. o f 90 x 1 0 6 , and d e f e c t i v e genom es

com posed e n t i r e l y o f r e i t e r a t i o n s o f th e te r m in a l r e p e a t

have been c h a r a c t e r i s e d .

CCV i s th e o n ly i d e n t i f i e d member o f group IV .

C housterm an e t a l . (1 9 7 9 ) p r e se n te d r e s t r i c t i o n maps o f CCV

DNA, show ing t h a t th e genome c o n s i s t s o f a u n iq u e r e g io n o f

62 x 1 0 in m .w t ., bounded on each s id e by a d i r e c t r e p e a t

o f 12 x 1 0 ^ . Ho m inor bands were d e t e c t e d and , u n l ik e IIVS

and ilVA, th e te r m in i were n o t h ig h ly r e i t e r a t e d .

The LEA o f two o th e r h e r p e s v ir u s e s has to some e x t e n t

been c h a r a c t e r i s e d , but d e te r m in a t io n o f t h e ir genome

s t r u c t u r e s lias been hampered by d i f f i c u l t i e s in c l o n a l l y

- 14 -

p u r i f y in g th e s e v ir u s e s from p o s s ib l e d e f e c t i v e v i r u s .

VZV DNA h as a m .w t. o f 8 0 -1 0 0 x 10 6 and r e s t r i c t i o n p r o f i l e s

o f s e v e r a l c l i n i c a l i s o l a t e s a re v er y s im i la r (O akes e t a l . ,

1 9 7 2 ; l i t i s e t a l . , 1977; Rapp e t a l . , 1 9 7 7 ; R ich a rd s e t a l . ,

1 979; Dumas e t a l . , 1 9 8 0 ) . Rapp e t a l . (1 9 7 7 ) f a i l e d to

d e t e c t r e p e a te d seq u e n c es by k i n e t i c h y b r id i s a t io n , but

subm olar r e s t r i c t i o n fra g m en ts have been d e t e c t e d .

IviDV DNA lias a m .w t. s im i la r to t h a t o f H SV -l, and subm olar

r e s t r i c t i o n fra g m en ts have been d e te c te d (Lee e t a l . , 1971;

B achenheim er e t a l . , 1 9 7 2 ; H ir a i e t a l . , 1 9 7 9 ) .

THE LYTIC CYCLE OP HERPES SIMPLEX VIRUS

I n t e r a c t io n o f v ir u s w ith th e c e l l

A fte r e n tr y o f HSV in t o th e p e r m is s iv e c e l l , th e DNA

e n t e r s th e n u c le u s , where i t i s t r a n s c r ib e d and th e mRNA

tr a n sp o r te d to th e c y to p la sm . M ost s y n t h e s is e d p o ly p e p t id e s

a re tr a n sp o r te d in t o th e n u c le u s where DNA r e p l i c a t i o n and

v ir u s a ssem b ly ta k e p la c e . In th e c o u r se o f i n f e c t i o n , HSV

i n h i b i t s h o s t c e l l m acrom olecu lar s y n t h e s i s . V iru s may

spread by r e l e a s e o f i n f e c t i o u s p a r t i c l e s from th e c e l l o r

by c e l l f u s i o n .

H erpes s im p lex v i r f o n s w i l l a d so rb to th e c e l l s u r fa c e

a t 4 ° , but p e n e t r a t io n r e q u ir e s a h ig h e r tem p era tu re

(Fam ham and Newton, 1959; Holmes and W atson, 1 9 63; Huang

and Wagner, 1 9 0 4 ) . The r o u te by w hich v i r i o n s e n te r th e

c e l l has r a is e d c o n s id e r a b le f u r o r e . I t was co n c lu d ed fronr

s e v e r a l e a r l i e r e le c t r o n m ic r o sc o p ic s t u d ie s t h a t th e v ir u s

i s ta k en up by p in o c y t o s i s in t o v a c u o le s (H olm es and W atson,

1963; ilummeler e t a l . , 1969; D a le s and S i lv e r b e r g , 1 9 6 9 ) .

On th e o th e r hand, morgan e t a l . (1 9 6 8 ) i n s i s t e d t h a t v ir u s

e n tr y o c c u r s by d i r e c t f u s io n w ith th e c e l l membrane.

- 13 -

S u b seq u en t work i s c o n s i s t e n t w ith th e p r e se n c e o f s p e c i f i c

v ir u s r e c e p t o r s on th e c e l l s u r fa c e (H ochberg and B eck er , 1968

B lom berg, 1979; Vahlne e t a l . , 1 9 7 9 ) . M oreover, e v id e n c e

t h a t v i r io n g ly c o p r o t e in s fu n c t io n in a d s o r p t io n and

p e n e t r a t io n makes d i r e c t f u s io n th e more t e n a b le H y p o th e s is

a t p r e s e n t (Cohen e t a l . , 1973; Sarm iento e t a l . * 1 9 7 9 ) .

The v i r i o n e n v e lo p e i s m ost p ro b a b ly r e q u ir e d f o r i n f e c t i v i t y

(S m ith , 1 9 64; S p r in g and Roizm an, 1 968; S t e in e t a l . , 1 9 7 0 ;

R u b en ste in e t a l . , 1 9 7 2 ) . A f te r e n tr y th e v i r io n s t r u c t u r e

i s degraded in th e cy to p la sm and th e DNA tr a n s p o r te d r a p id ly

by an unknown m echanism in t o th e n u c le u s (H ochberg and B eck er ,

1968; Hummeler e t a l . , 1 9 6 9 ) . Knipe e t a l . , (1 9 8 1 ) have

r e c e n t ly d e s c r ib e d a t s m utant w hich com plem ents o th e r t s

m u tan ts p o o r ly , and w hich e x p r e s s e s no p o ly p e p t id e s a t th e

n o n p e r m iss iv e te m p er a tu r e . The l e s i o n may map in a gen e

o o d in g a t l a t e t im e s i n i n f e c t i o n f o r a s t r u c t u r a l p r o t e in ,

and th e a u th o r s p ro p o se t h a t th e d e f e c t i s in u n c o a t in g th e

v i r u s .

I n f e c t io n o f c e l l s i n t i s s u e c u l t u r e w it h HSV r e s u l t s

i n c e s s a t i o n o f m it o s i s (S to k e r and N ew ton, 1 9 5 9 ; W ildy e t

a l . , 1 9 6 1 ) , and i n i t i a t i o n o f th e i n f e c t i o u s c y c l e i s

in d e p en d e n t o f th e c e l l u l a r phase o f DNA s y n t h e s is (Cohen

e t a l . , 1 9 7 1 ) . Soon a f t e r i n f e c t i o n , HSV c a u s e s an i n h i b i t i o n

o f c e l l DNA s y n t h e s is w hich i s d ep en d en t on p r o t e in s y n t h e s i s

(Roizman and R oane, 1 9 6 4 ) . The f a c t t h a t some HSV-2 t s

m u tan ts e i t h e r f a i l to i n h i b i t , o r s t im u la t e , h o s t c e l l DNA

s y n t h e s is a t th e n o n p e r m iss iv e tem p era tu re s u g g e s t s th a t

v ir u s f u n c t io n s are in v o lv e d in i n h i b i t i o n o f h o s t DNA

s y n t h e s i s (H a llib u r to n and Tim bury, 1973; Y am anishi e t a l . ,

1 9 7 9 ) . I t i s n o t known w h eth er th e i n h i b i t i o n i s a r e s u l t o f

i n h i b i t i o n o f c e l l RNA and p r o t e in s y n t h e s is (Roizman e t a l . ,

1 9 6 3 ) , r a th e r than a d i r e c t e f f e c t on DNA s y n t h e s i s .

- 16 -

I n h ib i t io n o f p r o te in s y n t h e s is in v o lv e s d is a g g r e g a t io n

o f h o s t p o ly r iu o so m e s and t h e i r r e a sse m b ly ( S y d is k is and

Roizman, 1 9 6 6 ) . The t o t a l p r o c e in s y n t h e s i s in g c a p a c it y

d e c r e a s e s th ro u g h o u t i n f e c t i o n , a f a c t w h ich S i l v e r s t e i n and

S n g e lh a rd t (1 9 7 9 ) in t e r p r e t e d a s th e a ccu m u la tio n o f non

f u n c t io n a l p o ly r ib o so m e s , Fenw ick and W alker (1 9 7 3 ) have

shown t h a t UV i r r a d ia t io n o f HSV-2 d o es n o t a f f e c t i n h i b i t i o n

o f h o s t c e l l m acrom olecu lar s y n t h e s i s , and co n c lu d ed t h a t

i n h i b i t i o n i s due to a s t r u c t u r a l com ponent o f th e v i r u s .

The same a u th o r s (1 9 7 9 ) have n o ted th e s p e c i f i c p h o sp h o r y la t io n

o f a h o s t r ib o so m u l p r o te in upon i n f e c t i o n , but co n c lu d ed

t h a t t h i s i s n o t th e ca u se o f i n h i b i t i o n o f h o s t p r o t e in

s y n t h e s i s . The HSV-2 fu n c t io n r e s p o n s ib le f o r i n h i b i t i o n h as

been mapped a t 0 .5 6 - 0 .5 7 f r a c t i o n a l genome u n i t s (Fenw ick

e t a l . , 1 9 79; H. 3 . M arsden, p e r s o n a l co m m u n ica tio n ).

IISV-1 i n f e c t i o n le a d s to th e e x p r e s s io n o f v i r u s -

s p e c i f i e d RNA, and c e l l RNA s y n t h e s i s i s r a p id ly su p p resse d

(Hay e t a l . , 1966; F la n a g a n , 1 9 6 7 ) . The p a r t i c u l a r l y ra p id

su p p r e s s io n o f rRNA s y n t h e s i s may be a t t r ib u t e d to two

in h ib i t o r y s t e p s (Wagner and Roizm an, 1 9 6 9 b ). F i r s t l y ,

th e r e i s a d e c r e a s e in th e r a t e o f s y n t h e s i s o f 45 S p r e c u r s o r

RNA, p erh a p s m ed iated by a v ir u s - in d u c e d i n h i b i t o r o f RNA

p o ly m era se I (S a sa k i e t a l . , 1 9 7 4 ) . S e c o n d ly , p r o c e s s in g o f

p r e c u r so r rRNA in t o m ature 18 S and 28 S rRNA i s i n h i b i t e d .

Talley-J3row n and G i l l e t t e (1 9 7 9 ) have n o te d ch a n g es in th e

p a t t e r n s o f sm a ll c y to p la s m ic RNA s y n t h e s i s e a r ly in i n f e c t i o n

l eroderm a pigm entosum c e l l s , ' in c lu d in g th e ap p earan ce o f

a n o v e l 380 n u c le o t id e s p e c i e s .

The a c t i v i t y o f KiJA p o ly m era se I I d e c l in e s on i n f e c t i o n ,

a f e a t u r e w hich P r e s to n and IJev/ton (1 9 7 6 ) a t t r ib u t e d n o t o n ly

to i n h i b i t i o n o f c e l l p r o te in s y n t h e s is but a l s o t o - a v i r u s -

in d u ced p o ly p e p t id e w hich »ay i n t e r a c t e i t h e r w ith th e

- 17 -

enzyme o r w ith th e ch rom atin com p lex . U s in g a c e l l l i n e

vh ich e x p r e s s e s a h ig h l e v e l o f g lo b in mRNA, N is h io k a and

S i l v e r s t e i n (1977 and 1 9 78) showed th a t HSV-l i n f e c t i o n

ca u ses d e g r a d a t io n o f p r e - e x i s t i n g c e l l mRNA. U n lik e

d i s s o c i a t i o n o f p o ly r ib o so m e s , t h i s i s d ep en d en t upon genome

e x x jress lo n . N e v e r t h e le s s , a low l e v e l o f c e l l mRNA i s

s y n th e s is e d even l a t e in i n f e c t i o n ( S t r in g e r e t a l . , 1 9 7 7 ) .

E n g lis and Newton (1 9 8 1 ) rexported t h a t HSV-l mRNA i s

t r a n s la t e d i n v i t r o w ith a lo w e r e f f i c i e n c y th an c e l l mRNA,

and s u g g e s te d t h a t , i f t h i s a p p l ie s in v i v o , i n h i b i t i o n o f

.lo s t c e l l RNA s y n t h e s i s would be in s tr u m e n ta l in e f f i c i e n t

v ir u s g ro w th .

C e l l chrom osom al b rea k a g es w hich o c c u r e a r ly in HSV

i n f e c t i o n a re th o u g h t to be due to a v ir u s - in d u c e d enzym e,

s in c e v ir u s DNA i s n o t i ) h y s ic a l ly a s s o c ia t e d w ith th e

b reakages (Waublce e t a l . , 1 9 6 8 ) . A lth ou gh chrom osom al

breakage seem s n o t to be cau sed by in t e g r a t io n o f v ir u s DNA

in to c e l l DNA, i t has been r e p o r te d th a t a b o u t 200 HSV genome

e q u iv a le n ts p er c e l l a re c o v a le n t ly a s s o c ia t e d w ith n o s t DNA

l a t e in i n f e c t i o n ( B ie g e le i s e n and Rush, 1976; B ie g e l e i s e n

3t a l . , 1 9 77; Y anagi e t a l . , 1 9 7 9 ) . The s ig n i f i c a n c e o f

th e se o b s e r v a t io n s in t h e . i n f e c t i o u s c y c le rem ain s to be s e e n .

The pathway by w hich v ir u s i s a ssem b led i n th e n u c le u s

i s p o o r ly u n d e r s to o d . E le c tr o n m ic r o sc o p ic s t u d ie s o f

n o rm a lly in f e c t e d c e l l s show c o n d e n sa t io n and m a rg in a tio n .o f

h o s t ch rom atin fo llo w e d by th e ap p earan ce o f c a p s id s o f n o ,

low o r h ig h e l e c t r o n - d e n s i t y , a s w e l l a s more b iz a r r e g r a n u la r ,

c r y s t a l l i n e , f i la m e n to u s and tu b u la r s t r u c t u r e s (Morgan e t a l . ,

1959; N i i e t a l . , 196 8 a ; Miyamoto and Morgan, 1971; Sm ith and de

H arven, 1 9 7 3 ) . o th e r p o t e n t i a l l y u s e f u l a t te m p ts to u n r a v e l

what i s u n d o u b ted ly a com plex p r o c e s s have in c lu d e d th e u se o f

~ 18 ~

m e ta b o lic i n h i b i t o r s (N ii e t a l . , 1 1 68b;Friedm ann e t a l . , 1975;

M arcian o-C ab ra l e t a l . , 19 / 7 ) , and s t u d i e s o f HSV-2 t s m utants

(C ab ra l and S c h a f fe r , 1976; A tk in son e t a l . , 1 9 7 0 ) . U n lik e

H SV -l, HSV-2 p ro d u ces tu b u la r s t r u c t u r e s in th e n u c le i o f

in f e c t e d c e l l s (Murphy e t a l • , 1 9 6 7 ) . T h is l a t e e v e n t seem s

n o t to r e q u ir e e n t ir e genome e x p r e s s io n , and k i n e t i c s t u d ie s

u s in g i n h i b i t o r s s u g g e s t t h a t su ch s t r u c t u r e s are in te r m e d ia te s

in v ir u s a ssem b ly r a th e r th an r e s u l t s o f a b er ra n t c a p s id

fo r m a tio n (Iw asak a e t a l . , 1979; Oda e t a l . , 1 9 7 9 ) .

H exagon al and p e n ta g o n a l in t r a n u c le a r c r y s t a l s o f c a p s id s have

f r e q u e n t ly been o b se rv ed (Morgan e t a l • , 1 9 5 8 ; M eln ick e t a l . ,

I 9 6 0 ) . I t i s n o t known how DNA i s e n c a p s id a te d in t o v i r i o n s ,

b u t P i g n a t t i and C a ssa i (1 9 8 0 ) i s o l a t e d n u c le o p r o te in co m p lex es

w hich in c lu d e d n o n -n u c leo so m a l DNA w ith c a p s id - l i k e s t r u c t u r e s

a t one te r m in u s . I t i s n o t c l e a r , h ow ever, w h eth er th e s e

r e p r e s e n t tr u e in te r m e d ia te s in th e e n c a p s id a t io n p r o c e s s ,

o r breakdown p r o d u c ts .

lidV i n f e c t i o n i s accom panied by e x t e n s iv e r e d u p l ic a t io n

o f th e n u c le a r membrane, a t w hich c a p s id s a c q u ir e e n v e lo p e s

a s th e y p a s s in t o th e p e r in u c le a r c i s t e m a (F a lk e e t a l • , 1959;

E p s te in , 1 9 6 2 b ;D a r lin g to n and M oss, 1968; N i i , 1 9 7 1 ) .

A tk in so n e t a l . (1 9 7 8 ) r e p o r te d t h a t en velop m en t may o ccu r

by cle novo s y n t h e s is o f membrane in th e n u c le u s . Envelopm ent

a t c y to p la s m ic membranes may o ccu r f o l lo w in g d i s i n t e g r a t i o n

o f th e n u c le u s . M ature v ir u s p a r t i c l e s l e a v e th e c e l l by

r e v e r s e p h a g o c y to s is (D a r lin g to n and M oss, 1 9 6 8 ) , o r v i a

c y to p la s m ic d u c ts (S ch w artz and Roizm an, 1 9 6 9 ) , o r by b oth

m echanism s ( N i i , 1 9 7 1 ) . O b se r v a tio n s o f v ir u s e g r e s s a re

d ep en d en t on c e l l ty p e (S c h le h o fe r e t a l . , 1 9 7 9 ) .

P r o d u c t iv e i n f e c t i o n k i l l s th e h o s t c e l l , and c e l l u l a r

com ponents le a k in t o th e medium (Y/agner and Roizm an, 1969b;

McCormick, 1 9 7 8 ) . V iru s sp rea d s from c e l l to c e l l v i a th e

- 19 -

iiedium, and a l s o by d i r e c t i n t e r c e l l u l a r .p a ssa g e (S to k e r , 1 9 5 8 ) .

V iId -ty p e v ir u s u s u a l ly c a u se s rou n d in g up o f in f e c t e d c e l l s ,

out some m utant s t r a in s ( s.vn) ca u se e x t e n s iv e c e l l f u s io n

and th e fo r m a tio n o f s y n c y t ia (Gray e t a l . , 1958; Iloggan and

Roizman, 1 9 5 9 ) . The r e c r u itm e n t o f c e l l s in t o s y n c y t ia seem s

la r g e ly d ep en d en t upon th e r e c r u i t in g r a th e r than th e r e c r u i t e d

c e l l (Lee and S p ea r , 1980; B z ik and P e r so n , 1 9 8 1 ) . C e l l

fu s io n i s d ep en d en t on v ir u s e x p r e s s io n , s in c e s e v e r a l

in h ib i t o r s o r UV i r r a d ia t io n a b o l i s h s y n c y t ia fo r m a tio n

(O’Donovan and Roizm an, 1961; K ou sou las e t a l . , 1 9 7 8 ) .

I n h ib it io n - by 2 -d e o x y -D -g lu c o se o r tu n ica m y c in s t r o n g ly

in d ic a t e s t h a t v ir u s g ly c o p r o t e in s a re in v o lv e d in c e l l f u s io n

(G a lla h e r e t a l . , 1973; K e l le r , 1 9 76; K now les and P e r so n , 1976;

P iz e r e t a l . , 1 9 8 0 ) . In d eed , u s in g reco m b in a n ts betw een a

v ir u s d e f e c t i v e i n g ly c o p r o t e in C (gC) and one w ith a t s l e s i o n

in g ly c o p r o t e in B (g B ), M a n serv ig i e t a l . , (1 9 7 7 ) co n c lu d ed

th a t gB i s a f u s io n prom oter and gC a f u s io n s u p p r e s s o r .

However, m u lt ip le v i r a l and c e l l u l a r f a c t o r s a r e in v o lv e d in

c e l l f u s io n (Yamamoto e t a l . , 1975; Yamamoto and K abuta, 1977;

Huyechan e t a l . , 1979; Lee and S p ea r , 1 9 8 0 ) .

DBA s y n t h e s is

kuch o f th e more c o n c lu s iv e work on h e r p e s v ir u s DNA

s y n t h e s is has been done w ith PRV, so th e f o l lo w in g d i s c u s s io n

in c lu d e s b oth HSV and PRV.

R ice e t a l . (1 9 7 6 ) r e p o r te d t h a t , soon a f t e r i n f e c t i o n ,

in p u t DNA becom es s p e c i f i c a l l y a s s o c ia t e d w ith a c e l l n u c le a r

p r o t e in . In c o n t r a s t , th ey d id n o t f in d nev/1 y s y n t h e s is e d DNA

in such a com p lex , and Du A s y t h e s i s d o es n o t in v o lv e th e

norm al c e l l n u c leo so m a l a p p a ra tu s ( k o u t t e t e t a l • , 1 9 79;

L ein bach and Summers, 1 9 8 0 ) . Jacob and Roizman (1 9 7 7 )

r ep o r te d th a t onl^ a sm a ll t r a c t io n o f p a r e n ta l DNA e n t e r s

- 20 -

th e r e p l i c a t i v e p o o l , w hereas th e m a jo r ity o f p a r e n ta l and

progeny PRV DNA can r e p l i c a t e (Jean and B en -P o r a t, 1976;

B en -P o ra t e t a l . , 1 9 7 6 a ) • D e n s i t y - s h i f t ex p er im e n ts have

shown th a t HSV and PRV DNA a re r e p l i c a t e d s e m ic o n s e r v a t iv e ly

(K aplan and B en -P o r a t, 1964; K o lb er , 1975; Khan e t a l . , 1 9 7 8 ) .

M ost s i n g l e s tra n d b reak ages in p a r e n ta l DNA a re r e p a ir e d p r io r

to DNA r e p l i c a t io n (B en -P o ra t e t a l . , 1 9 7 & d ),b u t n a s c e n t DNA

c o n t a in s s in g l e s tra n d ed r e g io n s and c o v a le n t ly l in k e d RNA

(B isw a l e t a l . , 1974; Murray and B is w a l, 1 9 7 4 ; H ir sc h e t a l . ,

1976; Sh lom ai e t a l . , 1976; M u ller e t a l . , 1 9 7 9 a ) . P u ls e -c h a s e

e x p e r im e n ts in d ic a t e th a t new ly s y n t h e s is e d DNA se d im e n ts

a s la r g e h e te r o g e n e o u s m o le c u le s , and i s r a p id ly p r o c e s s e d

to a form sed irtien tin g w ith th e p r o p e r t ie s o f u n i t - l e n g t h

m o le c u le s ( h ir s c h e t a l . , 1976; Sh lom ai e t a l . , 1 9 7 6 ; Jacob

and Roizm an, 1 9 7 7 ; B en -P o ra t and T ok azew sk i, 1 9 7 7 ) . A s in g le

round o f PRV DNA r e p l i c a t io n ta k e s 25 o r 45 m in, d ep en d in g

on w hether r e p l i c a t io n i s u n i - or b i - d i r e c t i o n a l (B en -P o ra t-

e t a l . , 1 9 7 7 ) . Most sy ste m s f o r in v i t r o DNA s y n t h e s i s a l lo w

c o m p le t io n o f n a sc e n t DNA but n o t r e i n i t i a t i o n ( B e l l , 1 974;

B is w a l and Murray, 1974; Shlom ai and B eck er , 1975; B o ld en e t

a l . , 1975; K o lb er , 1975; B eck er and A sh er, 1 9 7 5 ; Sh lom ai e t

a l . , 1977; J o n g en ee l and B achenheim er, 1 9 8 0 ) . P r o m is in g

a tte m p ts have been made to e x t r a c t th e p h y s io lo g ic a l

r e p l i c a t i o n com plex from in f e c t e d c e l l n u c le i ( P ig n a t t i e t

a l . , 1979 ; K nopf, 1 9 7 9 ) .

T e m p e r a tu r e -s e n s it iv e m u tan ts in ab o u t h a l f o f th e

i d e n t i f i e d com p lem en ta tion grou p s o f HSV and PRV a re d e f i c i e n t

i n DNA s y n t h e s is to a g r e a te r or l e s s e r e x t e n t , and t h e r e fo r e

i t i s l i k e l y th a t .many gene p ro d u cts a re in v o lv e d i n t h i s

p r o c e s s , in c lu d in g th e v ir u s -c o d e d DNA p o ly m era se (H a ll ib u r to n

and Tim bury, 1973 and 1 9 7 6 ; P r in g le e t a l . , 1973; S c h a f fe r

e t a l . . 1973; iSsparza e t a l . , 1974; D en yesh -M eln ick e t g l . » 1 9 7 4 ;

P u r ifo y and B e n y e s n -^ e ln ic k , 1975; Aron e t a l . » 1975; C ourtney

e t a l . , 1 9 76; Bone e t a l . , 1 9 7 8 ) .

e l e c t r o n m ic r o sc o p ic ex a m in a tio n o f r e p l i c a t in g DNA

h a s r e v e a le d l i n e a r m o le c u le s s m a lle r th a n , e q u a l in s i z e t o ,

o r la r g e r than v ir io n DNA, c i r c l e s o f u n i t s i z e o r s m a lle r ,

and la r g e ta n g le d m o le c u le s . Such m o le c u le s may c o n ta in

s i n g l e s tr a n d ed r e g io n s , f o r k s , e y e s , lo o p s o r c o l la p s e d

r e g io n s (S h lom ai e t a l . , 1976; Jean and B e n -P o r a t, 1976;

B en -P o r a t e t a l . , 1 976b ;H ixon , 1977; Jean e t a l . , 1977;

H ir sc h e t a l . , 1977; Priedraann e t a l . , 1 9 77; Jacob and Roizm an,

1 9 7 7 ) . The f o l lo w in g r e p l i c a t io n m odel h a s been d e r iv e d from

th e s e and o th e r d a ta . O b se r v a tio n s th a t HSV-l DNA i s

t e r m in a l ly red u n d a n t, th a t te r m in a l r e s t r i c t i o n fra g m en ts

o f IiSV-1 DNA a re red u ced in abundance in in f e c t e d c e l l n u c le i

(J a co b e t a l • , 1 9 7 9 ) , th a t a p r o p o r t io n o f in p u t PRV DNA

a c q u ir e s s in g l e s tra n d ed ends (Jean and B e n -P o r a t, 1976) and

a p r o p o r t io n becom es c i r c u l a r , and t h a t PRV DNA i n th e f i r s t

round o f r e p l i c a t io n i s c i r c u la r (B en -P o ra t and V each, 1 9 8 0 ) ,

a re in su p p o rt o f th e h y p o th e s is t h a t l i n e a r in p u t DNA

c i r c u l a r i s e s a l t e r i n f e c t i o n , p ro b a b ly in v o lv in g th e a c t io n

o f an e x o n u c le a s e . H e a d - t o - t a i l co n ca tem ers a re th en form ed

by an unknown m echanism , but w hich may be a r o l l i n g c i r c l e

(Jean and B en -P o r a t, 1976; Jean e t a l • , 1 9 77; B en -P o ra t and

T ok azew sk i, 1977; B en -P o ra t and R ixon , 1 979; Jacob e t a l . , 1 9 7 9 ) .

The q u e s t io n o f how u n i t - l e n g t h DNA m o le c u le s r e s u l t from

c o n ca to m c rs , w ith fo u r eq u im olar arran gem en ts o f 1 and S,

i s c o n s id e r e d in th e D is c u s s io n . M odels f o r DNA s y n t h e s i s

in v o lv in g in d ep en d en t r e p l i c a t io n o f L and S ( Skare and Summers,

1 9 7 7 ) o r r e p l i c a t i o n o f l i n e a r m o le c u le s from a te r m in a l

h a ir p in lo o p (R ixon , 197'/; Jacob and Roizm an, 1 977) f a i l e d to

f in d su b seq u en t su p p o r t . B en -P orat and Kaplan (1 9 6 3 ) showed

- 22 -

th a t a lth o u g h a m in o r ity o f m ature PRV DNA i s e neap s i d a te d ,

i t i s s e l e c t e d a t random from th e p o o l . S im i la r ly a m in o r ity

o f HSV-l DNA i s e n c a p s id a te d ( R u s s e l l e t a l . , 1 9 6 4 ) , and

P r en k e l and Roizman (1 9 7 2 a ) co n c lu d ed t h a t m o le c u le s w ith

fe w er n ic k s a re p r e f e r e n t i a l l y p ack aged , a lth o u g h i t i s e q u a lly

l i k e l y t h a t n ic k s a r e r e p a ir e d d u r in g p a c k a g in g .

Two ap p ro a ch es have in d ic a te d t h a t HSV-l and PRV DNA

c o n ta in an o r ig in o f r e p l i c a t io n in th e r e g io n o f th e S

term in u s , and p erh ap s a n o th e r i n U- . Sh lom ai e t a l . (1 9 7 6 )

and Priodmann e t aJ_. ( 1 9 7 7 ) d e sc r ib e d two o r ig in s o f

r e p l i c a t io n from e le c t r o n m ic r o sc o p ic s t u d ie s o f r e p l i c a t in g

iiSV-1 DMA, one c o r r e sp o n d in g to TR and IR0 , and th e o th e r to

a p o s i t i o n 40# from one te r m in u s . H ir sc h e t a l . (1 9 7 7 )

d e te c te d g r e a te r in c o r j jo r a t io n o f i s o t o p e in a p u lse d l a b e l

in r e s t r i c t i o n fra g m en ts c o n ta in in g th e j o i n t r e g io n .

Jean e t a l . (1 9 7 7 ) o b ta in e d r e s u l t s s im i la r to th o s e

d e s c r ib e d above from t h e i r e le c t r o n m ic r o sc o p ic a n a ly s i s

o f r e p l i c a t in g PRV HR A, a lth o u g h 1 0 # o f m o le c u le s w ith4

r e p l i c a t io n e y e s a p p a r e n t ly e x h ib it e d m u lt ip le r e p l i c a t io n

s i t e s . H en-P orat and Veuch (1 9 8 0 ) co n c lu d ed from b lo t

h y b r id is a t io n o f PRV nil A th o u g h t to have been i s o t o p i c a l l y

l a b e l l e d d u r in g th e f i r s t round o f r e p l i c a t i o n t h a t th e main

o r ig in o f r e p l i c a t io n i s a t o r n ea r th e S te r m in u s , from

w hich DliA s y n t h e s is p ro ce ed s u n i d i r e c t i o n a l l y . They su g g e s te d

th a t th e o th e r o r ig in 40# from one term in u s i s u sed r e l a t i v e l y

in f r e q u e n t ly d u r in g th e f i r s t round o f r e p l i c a t i o n .

As a secon u ap p roach , s t r u c t u r a l a n a ly s i s o f d e f e c t iv e

v ir u s genomes h as p ro v id ed some e v id e n c e f o r th e l o c a t io n s

o f o r ig in s o f J)AA r e p l i c a t i o n . High m u l t i p l i c i t y p a ssa g e

o f HSV or PRV r e s u l t s in a c y c l i c p a t te r n o f p a r t i c l e to '

i x . f e c t i v i t y r a t i o end o f te n tn e ap p earan ce o f DNA o f n o v e l

B en -P o ra t e t a l . , 1974; hu rray e t a l * , 1 9 75; Stegmari e t a l . ,

1 9 7 8 ) . DNA from th e d e f e c t i v e i n t e r f e r i n g p a r t i c l e s i s

a p p ro x im a te ly th e same s i z e a s , hut h as a lo w e r k i n e t i c

c o m p le x ity th a n , sta n d a rd DNA (V/agner e t a l . , 1974; Bookout

e t a l . , 1 9 7 9 ) . P a r t i a l d e n a tu r a t io n m apping has shown th a t

d e f e c t i v e DNA c o n s i s t s o f m u lt ip le h e a d - t o - t a i l r e i t e r a t i o n s

o f a b a s ic u n i t ( liu b e n s te in and K aplan, 1 9 7 5 ; P ren k e l e t a l . t

1 9 7 5 ) .

Two ty p e s o f rej^eat u n i t have been d e s c r ib e d from

r e s t r i c t i o n e n d o n u c le a se a n a ly s i s o f HSV-l d e f e c t i v e DNA

(K aerner e t a l • , 1979; P r en k e l e t a l . , 1 9 8 0 ) . That o f c l a s s I

c o n t a in s axjp rox im ately 8 .5 kbp o f DNA c o m p r is in g TR and an

a d ja c e n t p o r t io n o f . (P re n k e l e t . a l . , 1 9 7 5 , 1976 and 1980;

lo c k e r and P r e n x e l, 1978 and 1979a;Graham e t a l . , 1978;

V lazny and P r e n k e l, 1 9 8 1 ) . Y/hereas one end o f th e d e f e c t iv e

genome i s u n iq u e and p ro b a b ly p o s s e s s e s an a se q u e n c e , th e

o th e r term in u s i s th o u g h t to be random in n a tu r e . Such a

s t r u c t u r e s u g g e s t s t h a t m a tu ra tio n and p a ck a g in g o f DNA

o ccu r by a s p e c i f i c r e c o g n it io n o f th e S term in u s and an

ap p rox im ate le n g th m easurem ent• T h is i s u n l ik e ly to be th e

c a s e f o r m a tu r a tio n o f sta n d a rd IISV-1 DNA b eca u se both

te r m in i a re u n iq u e . I t i s n o t y e t c l e a r w h eth er th e a

seq u en ce i s p r e s e n t in ea ch r e p e a t u n i t o f c l a s s I d e f e c t i v e

genom es. S iz e h e t e r o g e n e i ty o f th e r e p e a t u n i t in d i f f e r e n t

d e f e c t i v e genom es o f t n i s c l a s s i s due to v a r ia t io n in th e

le n g tn o f se q u e n c e s in c lu d e d t h e r e in . The k i n e t i c s o f

d e f e c t i v e DNA s y n t a c s i s , i t s s e n s i t i v i t y to th e v ir u s DNA■> •»

p o lym erase i n h i b i t o r p h o s if io n o a c e t ic a c id (PAA), and th e

f a c t t h a t c o i n i c c l i o n v /itn se p a r a te d r e jje a t u n i t s generates

- R4 -

f u l l le n g th d e f e c t i v e genom es, argue t h a t d e f e c t i v e DNA

c o n ta in s th e IISV-1 r e p l i c a t io n o r ig in w hich i s f u n c t io n a l

in th e r e p l i c a t i o n o f d e f e c t i v e DNA (P r e n k e l e t a l . , 1980;

V lazny and P r e n k e l, 1 9 8 1 ) . The o b s e r v a t io n t h a t s in g l e

d e f e c t i v e DNA m o le c u le s c o n ta in o n ly one 3 i s e o f r e p e a t u n it

makes i t t e n a b le th a t th ey a re s y n t h e s is e d by a r o l l i n g c i r c l e

m echanism from a c i r c u l a r r e p e a t u n i t . In su p p o r t o f t h i s

h y p o th e s is , B ecker e t a l . (1 9 7 8 ) o b se rv ed c i r c u l a r and

c i r c u l a r - l i n e a r m o le c u le s o f v a r io u s s i z e s by e le c t r o n

m icro sco p y o f r e j iL ic a t in g d e f e c t i v e DNA.

The r e p e a t u n i t o f c l a s s I I d e f e c t i v e DNA c o n s i s t s o f

th e S te r m in a l r e g io n l in k e d to a r e g io n o f m apping a t

0 .3 4 - 0 .4 0 f r a c t i o n a l genome u n i t s . The g r e a t e r s e n s i t i v i t y

o f t h i s c l a s s than c l a s s I to PAA su p p o r ts th e p r e se n c e o f a

s e p a r a te r e p l i c a t io n o r ig in in U^. In d ee d , i t h as been n o ted

th a t a t te m p ts to c lo n e in t o p la sm id s fra g m en ts from t h i s

r e g io n o f have c o n s i s t e n t l y le d to th e d e l e t io n o f ab ou t

100 bp a t a s p e c i f i c l o c a t io n , p erh ap s i n d ic a t in g th e

p r e se n c e o f an in v e r te d r e p e a t in v ir u s DNA w hich p r o v e s

u n s ta b le in p la sm id s (P o s t e t a l . , 1 9 80; B. M atz, p e r s o n a l

co m m u n ica tio n ).

RNA s y n t h e s is

iiSV-1 RNA s y n t h e s is i s a s s e n s i t i v e a s c e l l RNA s y n t h e s i s

to i n h i b i t i o n by *>c-amani t i n , an i n h i b i t o r o f RNA p o ly m era se I I

(A lv/ine e t a l . , 1974; B en-Z eev e t a l . , 1 9 7 6 ) , and a l l s t a g e s

o f v i r a l t r a n s c r ip t io n a re e q u a lly s e n s i t i v e (C o sta n zo e t a l • ,

1 9 7 7 ) . m oreover, u3V~l RNA s y n t h e s is i s r e s i s t a n t to th e

urug in c e l l s p o s s e s s in g an cx’-a m a n it in r e s i s t a n t RNA

p o lym erase I I (C ostan zo e t a l . , 1 9 7 7 ; B en-Z eev and B eck er ,

1 9 7 7 ) . Lowe (1 8 7 8 ) was u n ab le to d e t e c t RNA p o ly m era se

a c t i v i t i e s in th e in x ecteu . c e l l o th e r than th o s e n o rm a lly

- 25 -

p r e s e n t in the u n in fe c te d c e l l . These o b s e r v a t io n s s t r o n g ly

in d ic a t e t h a t c e l l RNA p o lym erase I I i s r e s p o n s ib le f o r

IiSV-1 RNA s y n t h e s i s a t a l l s ta g e s o f i n f e c t i o n , a lth o u g h

m o d if ic a t io n o f th e t r a n s c r ip t io n a l s p e c i f i c i t y o f t h i s

enzyme d u r in g i n f e c t i o n i s n o t r u le d o u t .

HSV-l mRNAs a re p o ly a d e n y la te d a f t e r t r a n s c r ip t io n

(B aclienheim er and Roizm an, 1 9 7 2 ) . I t h a s been r e p o r te d th a t

a s i g n i f i c a n t p r o p o r t io n o f mRNA i s n o t p o ly a d e n y la te d

( S i l v e r s t e i n e t a l . , 1 9 7 3 ; S tr in g e r et; a l . , 1 9 7 7 ) , but

S i l v c r s t e i n e t a l . (1 9 7 6 ) showed t h a t m ost o r a l l v ir u s

mRNAs a re p o ly a d e n y la te d , i n g e n e r a l p o s s e s s in g 3 0 , 50 o r

155 a d e n y lic a c id r e s id u e s a t th e 3 1 te r m in u s . The

c o m p a r tm e n ta lisa t io n o f s h o r te r p o ly a d e n y lic a c id t r a c t s in

th e n u c le u s , and th e lo n g e r in th e c y to p la sm , was in t e r p r e t e d

by th e l a t t e r a u th o r s a s two s e p a r a te p r o c e s s in g s t e p s in

p o ly a d e n y la t io n . HSV-l mRNAs a l s o p o s s e s s a 7 -m e th y lg u a n o s in e

5 ' - t r ip h o s p h a t e cap and i n t e r n a l l y m eth y la ted n u c le o t id e s

(B a r tk o sk i and Roizm an, 1976; Moss e t a l . , 1977)* Jacquem ont

and H uppert (1 9 7 7 ) showed t h a t HSV-l r e p l i c a t i o n i s r e v e r s ib ly

b lo ck ed by 5 f - S - i s o b u t y la d e n o s in e , an i n h i b i t o r o f m e th y la t io n ,

e s p e c i a l l y o f th e c a p . Assum ing t h a t t h i s i s th e s i g n i f i c a n t

in h ib i t o r y a c t io n o f t h e ‘d ru g , t h i s r e s u l t s u g g e s t s t h a t

m e th y la t io n i s e s s e n t i a l f o r t r a n s la t io n o f mRNA. C e s s a t io n

o f m e th y la t io n o f in t e r n a l n u c le o t id e s a t l a t e t im e s may be

due to e x p r e s s io n o f an e a r ly gene (B a r tk o sk i and Roizm an, 1 9 7 8 ) .

HSV t r a n s c r ip t io n i s a com plex p r o c e s s under t ig h t

tem p ora l c o n t r o l . H arly work u s in g l iq u id h y b r id is a t io n showed

t h a t m ost o f th e H3V-1 genome i s e x p r e sse d a t e a r ly (b e fo r e

th e o n s e t o f DNA s y n t h e s i s ) and l a t e t im e s (P r e n k e l and

Roizm an, 1972b;W agner, 1972; P ren k e l e t a l . , 1973; Swanstrom

anu Wagner, 1974; S tr in g e r e t a l . , 1 9 7 7 ) . B a r ly mRNA

c o m p r ise s a s u b s e t o f l a t e mRNA, and ea ch group c o n s i s t s o f

s c a r c e and abundant t r a n s c r ip t s , Wagner e t a l . (1 9 7 2 ) p rop osed

a v e r y e a r ly c l a s s w hich form s a s u b s e t o f e a r ly s p e c i e s .

The e v id e n c e f o r th r e e m ajor c l a s s e s o f t r a n s c r ip t s was

su p p o rted by ex p e r im e n ts in v o lv in g th e u se o f m e ta b o lic

i n h i b i t o r s . I n i t i a l l y i t was r e p o r te d t h a t mRNA made i n th e

a b se n c e o f de novo p r o t e in s y n t h e s i s was s im i la r to t h a t made

a t e a r ly t im e s (P r e n k e l e t a l . , 1973; Swanstrom e t a l . , 1 9 7 5 ) ,

but i t soon became c l e a r th a t im m ediate e a r ly (IE ) mRNA

produced in th e p r e se n c e o f c y c lo h e x im id e form s a more

r e s t r i c t e d c l a s s (Kozak and Roizm an, 1 9 7 4 ; Roizman e t a l . , 1 9 7 4 ) .

I n h ib i t io n o f DNA s y n t h e s is c a u s e s th e p r o d u c t io n o f mRNA

v e r y s im i la r to t h a t made a t e a r ly t im e s (Wagner e t a l . , 1972;

Murray e t a l . , 1974; Swanstrom and Wagner, 1974; Swanstrom

e t a l . , 1 9 7 5 ) .

H y b r id is a t io n o f IE mRNA to n i t r o c e l l u l o s e s t r i p s

c o n t a in in g r e s t r i c t i o n fra g m en ts o f HSV-l DNA a llo w e d IE g e n e s

to be mapped on th e genome (C lem en ts e t a l . , 1977; Jon es

e t a l . , 1 9 7 7 ) . The d i r e c t i o n s o f t r a n s c r ip t io n o f t h e s e

n o n -c o n t ig u o u s g en es were e s t a b l i s h e d by h y b r id is a t io n u s in g

s h o r t cDNA s y n t h e s is e d u s in g p o ly a d e n y la te d mRNA (C lem en ts

e t a l . , 1979; Anderson e t ' a l . , 1 9 8 0 a ;J . B. C lem en ts , p e r s o n a l

co m m u n ica tio n ). I s o l a t io n o f mRNAs from m ethylraercui*ic

h y d ro x id e a g a r o se g e l s fo llo w e d by t h e ir t r a n s l a t io n i n v i t r o

h as a llo w e d th e c o r r e la t io n o f mRNA s i z e , genome l o c a t io n ,

and p o ly p e p t id e s i z e (W atson e t a l . , 1979; T a lley -B ro w n and

M i l l e t t e , 1979; Anderson e t a l . , 1 9 8 0 a ) . C lem en ts e t a l . (1 9 7 9 )

p rop osed a m odel f o r t r a n s c r ip t io n o f IE g e n e s u t i l i s i n g a

s in g l e prom oter in THcj/IRg, but- su b seq u en t work h as shown

th a t each IE gene has i t s own prom oter (E a sto n and C lem en ts ,

1980; h i l l e t t e and iCLaiber, 1580; Mackem and Roizm an, 1980;

- 27 -

H. J . R ixon , p e r s o n a l co m m u n ica tio n ). D ata c o n c e r n in g th e

HSV-l ID g e n es are summarised in P ig u re A 1 .4 . A lth ough

P r en k e l e t a l . (1 973) reported , th a t th e t r a n s c r ip t io n a l

program o f HSV-2 i s s i g n i f i c a n t l y d i f f e r e n t from t h a t o f

H SV -l, H aston and C lem en ts (1 9 8 0 ) have c l e a r l y shown by

b lo t h y b r id is a t io n , mRNA i s o l a t i o n and in v i t r o t r a n s la t io n

t h a t th e two v ir u s e s sh a re v er y s im i la r IE t r a n s c r ip t io n p a t t e r n s .

H arly mRNA form s a more com plex c l a s s mapping th ro u g h o u t

th e HSV-l genome (C lem en ts e t a l . , 1977; S tr in g e r e t a l . , 1 9 7 8 ) .

Some 16 s p e c ie s have been mapped, m ost o f w hich se e m in g ly

have in d iv id u a l p rom oters (H o lla n d e t a l . , 1 979; M i l l e t t e and

K la ib e r , 1 9 8 0 ) . Anderson e t a l . (1 9 7 9 ) lo c a t e d on th e genome

a p p r o x im a te ly 40 l a t e mRNAs r a n g in g i n s i z e from 1 .5 to

g r e a t e r th a n 8 kb . C o sta e t a l . (1 9 8 1 ) have mapped th e

m ajor l a t e mRNA, w hich i s p ro b a b ly n o t s p l i c e d and c o d e s fo r

th e m ajor c a p s id p r o te in o f m .w t. 1 5 5 ,0 0 0 . I t i 3 o b v io u s

t h a t d i s s e c t i n g tn e c o m p le x it ie s o f th e s e t r a n s c r ip t io n a l

p a t t e r n s w i l l occupy i n v e s t i g a t o r s f o r n o t a few y e a r s .

R egard in g th e tem p ora l c o n t r o l o f th e t r a n s c r ip t io n a l

program o f H SV -l, Y/ard and S te v e n s (1 9 7 5 ) showed th a t when

t r a n s c r ip t io n was b lo ck ed by a c tin o m y c in D, e a r l i e r mRNAs

ten d ed to be more s h o r t - l iv e d th an l a t e r o n e s , s u g g e s t in g

a c o n t r o l mechanism o p e r a t in g a t t h i s l e v e l . A seco n d ty p e

o f c o n t r o l , a c t in g a t th e l e v e l o f p r o c e s s in g o f i n i t i a l

t r a n s c r ip t s in th e n u c le u s and t r a n s lo c a t io n o f m ature mRNAs

to th e c y to p la sm , i s l i k e l y to be o p e r a t iv e e s p e c i a l l y in

l a t e r s t a g e s o f i n f e c t i o n . T r a n s c r ip ts i s o l a t e d from n u c le i

a t l a t e t im e s a re la r g e r than c y to p la s m ic mRNAs, and some

n u c le a r s p e c ie s are u n d e r -r e p r e se n te d in th e cy to p la sm

(iVagner ana Roizman, 1969a and b; Wagner, 1972; Kozak and

- 28 -

Roizman, 1974-; C lem ents e t a l . , 1977; J o n es and Roizm an, 1979;

Jacquem ont e t a l . , 1 9 8 0 ) . However, s im i la r c la im s th a t m ost

o r a l l o f th e genome i s t r a n s c r ib e d in th e n u c le u s under IE

c o n d i t io n s , and t h a t m ost t r a n s c r ip t s a ccu m u la te in th e n u c le u s

so th a t o n ly c e r t a in s p e c ie s e n te r th e cy to p la sm (Kozak and

Roizm an, 1974; J o n es and Roizm an, 1 9 79) have been c o u n tered

by o th e r s who showed t h a t n u c le a r and c y to p la s m ic IE RNAs a re

o f s im i la r c o m p le x it ie s (C lem en ts e t a l . , 1 9 77; E aston and

C lem en ts, 1 9 8 0 ; A nderson e t a l . , 1 9 8 0 a ) . l i q u i d h y b r id is a t io n

s t u d ie s showed t h a t a s i g n i f i c a n t p r o p o r t io n o f th e genome i s

sy m m e tr ic a lly t r a n s c r ib e d a t l a t e t im e s (B en -Z eev and Becker",

1975; Jacquem ont and Roizm an, 1975a and b; Kozak and Roizm an,

1975; M iov ic and P iz e r , 1 9 8 0 ) .

The m ost im p o rta n t l e v e l o f c o n t r o l i s th o u g h t to be

t h a t o f t r a n s c r ip t io n a l i n i t i a t i o n . The HSV-l m u tan ts

tsK , tsD and t s T have in d ep en d en t l e s i o n s i n th e gene c o d in g

f o r IE mRNA 3 , w hich i s t r a n s la t e d in t o th e IE p o ly p e p t id e

Vmw IE 175 (IC P4) (P r e s to n e t a l . , 1978; Stow and W ilk ie ,

1978; P r e s to n , in p r e s s ) . A lth ou gh t s K com plem ents tsD and

t s T in some t e s t s (C rom bie, 1 9 7 5 ) , i t i s now o v erw h elm in g ly

l i k e l y t h a t th e th r e e l e s i o n s a r e i n one g e n e . The m u ta tio n

in tsK i s due to a s in g le , b ase p a ir change r e s u l t i n g in th e

rep la cem en t o f an a la n in e by a v a l in e r e s id u e (M -J. M urchie,

p e r s o n a l co m m u n ica tio n ). T hese m u tan ts p rod u ce a t non

p e r m is s iv e tem p era tu re mRNA and p o ly p e p t id e p r o f i l e s more

or l e s s s im i la r to th e IE p a t te r n s (M arsden e t a l . , 1976;

Watson and C lem en ts , 1 9 7 7 , 1978 and 1980; D ixon and S c h a f fe r ,

1 9 8 0 ) . The p r o p e r t ie s o f t h e s e m u tan ts show t h a t a t l e a s t

one IE f u n c t io n ( i . e . Vmw IE 175) i s c o n t in u o u s ly r e q u ir e d

f o r th e t r a n s i t i o n from IE to e a r ly t r a n s c r ip t io n a l p a t t e r n s .

P r e s to n (1 9 7 9 ) showed th a t t h i s fu n c t io n i s d i r e c t l y in v o lv e d

in th e a c t i v a t i o n a t th e t r a n s c r ip t io n a l l e v e l o f an e a r ly

- 29 -

gen e (d eo x y p y r im id in e k in a s e , dPyK). I n h i b i t i o n o f DNA

s y n t h e s is le a d s to an e a r ly t r a n s c r ip t io n a l p a t te r n and nuch

red u ced p r o d u c t io n o f l a t e p o ly p e p t id e s ( lio n e s s and Roizm an,

1974; H oness and W atson, 1 9 7 7 a ;C lem en ts e t a l . , 1977;

Watson and C lem en ts , 1 9 7 7 ; H olland e t a l . , 1 9 8 0 ) . A lth ough

W olf and Roizman (1 9 7 8 ) in t e r p r e t e d t h i s a s a req u irem en t

f o r both p a r e n ta l and p rogeny DNA te m p la te s f o r a norm al

l e v e l o f l a t e p o ly p e p t id e s y n t h e s i s , i t seem s more l i k e l y

th a t v ir u s DNA s y n t h e s is i s r e q u ir e d f o r th e t r a n s i t i o n from

e a r ly to l a t e p a t te r n s o f t r a n s c r ip t io n .

P r o te in s y n t h e s i s and m o d if ic a t io n

A p p rox im ately 50 HSV-induced p o ly p e p t id e s have been

d e t e c t e d i n in f e c t e d c e l l s by S D S -p o ly a cry la m id e g e l

e l e c t r o p h o r e s i s (Honess- and Roizm an, 1 973; M arsden e t a l . ,

1 9 7 6 ) . T w o-d im en sion a l g e l e l e c t r o p h o r e s i s h a s a llo w e d

250 p o ly p e p t id e s to be d i s t in g u i s h e d , a lth o u g h u n d o u b ted ly

many o f t h e s e r e s u l t from p o s t - t r a n s l a t i o n a l m o d if ic a t io n o f

p rim ary t r a n s l a t io n p r o d u c ts (H aarr and M arsden, 1 9 8 1 ) .

V iru s p r o t e in s a r e s y n t h e s is e d in th e cy to p la sm and

m o st, w ith th e e x c e p t io n o f g ly c o s y la t e d p o ly p e p t id e s , a re

th en tr a n sp o r te d to th e n u c le u s ( S y d is k is and Roizm an, 1968;

S p ear and Roizm an, 1968 and 1 9 7 0 ) . I n i t i a l s t u d ie s o f th e

k i n e t i c s o f s y n t h e s is o f v ir u s - in d u c e d p o ly p e p t id e s

d em o n stra ted tem p ora l c o n t r o l o f p r o t e in s y n t h e s i s , and c o n t r o l

o f p r o t e in abundance (H oness and Roizm an, 1 9 7 3 ) . E nzym atic

f u n c t io n s ten d to be produced e a r ly i n i n f e c t i o n , and m ost

s t r u c t u r a l p r o t e in s l a t e r ( R u s s e l l e t a l . , 1 9 6 4 )* The

su b seq u en t u se o f m e ta b o lic i n h i b i t o r s su g g e s te d th e e x i s t e n c e

o f th r e e k i n e t i c c l a s s e s o f p o ly p e p t id e s , a , and y% w hich

co rresp o n d in g e n e r a l to th e IE , e a r ly and l a t e mRNA s p e c ie s

(H on ess and Roizman, 1974 and 1 9 7 5 a ) . oi p o ly p e p t id e s are

- 30 -

s y n t h e s is e d im m ed ia te ly a l t e r r e l e a s e o f a c y c lo h e x im id e b lo ck

a t l e a s t one o f t h i s c l a s s i s r e q u ir e d f o r th e p r o d u c t io n o f

/5 p o ly p e p t id e s , f t f u n c t io n s a re r e q u ir e d f o r th e i n h i b i t i o n

o f <k p o ly p e p t id e s y n t h e s i s and th e s t im u la t io n o f y

p o ly p e p t id e s , w h ich in tu rn i n h i b i t s y n t h e s i s .

The t h r e e f o ld c l a s s i f i c a t i o n o f v ir u s - in d u c e d p o ly p e p t id e s

i s a s i m p l i s t i c m odel, s in c e P e r e ir a e t a l . (1 9 7 7 ) i d e n t i f i e d

two c l a s s e s o f j$ p o ly p e p t id e (^q and y#^), ea ch o f w h ich i s

p ro b a b ly d ep en d en t upon an c* f u n c t io n f o r e x p r e s s io n

(P r e s to n , 1 9 7 9 ) . S tu d ie s o f p o ly p e p t id e s y n t h e s i s in th e

p r e s e n c e o f an i n h i b i t o r o f DNA s y n t h e s i s have le d to th e

i d e n t i f i c a t i o n o f a t l e a s t two y c l a s s e s (H on ess and W atson,

1 9 7 7 a ;P ed erso n e t a l . , 1 9 8 1 ) . The la t t e r " a u t h o r s d e f in e d

s i x c l a s s e s o f p o ly p e p t id e on th e b a s i s o f t h e i r e x p r e s s io n

in th e p r e se n c e o f an i n h i b i t o r o f DNA s y n t h e s i s , and M arsden

e t a l . (1 9 7 6 ) d e s c r ib e d n in e c l a s s e s o f d ep en d en ce r e l a t i o n s h i p

b etw een p o ly p e p t id e s y n t h e s is and th e DNA p h en otyp e o f HSV-l

t s m u ta n ts . The p r o p e r t ie s o f a dom inant l e t h a l e a r ly

m utant i s o l a t e d by J o fr e e t a l . (1 9 8 1 ) a r e in su p p o r t o f th e

e x i s t e n c e o f a s l e a s t two fi and y c l a s s e s . P o s t e t a l . (1 9 8 1 )

e s t a b l i s h e d a c e l l l i n e c o n ta in in g a f t gen e IdPyK) under

oc (Vmw IK 1 7 5 ) t r a n s c r ip t io n a l c o n t r o l and fou n d t h a t dPyK

a c t i v i t y was in d u c e d by i n f e c t i o n w ith dPyK v i r u s . T h is*

e f f e c t was shown n o t to be due to a v ir u s oC f u n c t io n , so

th e y in t e r p r e t e d i t a s th e s t im u la t io n o f t r a n s c r ip t io n from

th e cx prom oter by a v i r io n s t r u c t u r a l p o ly p e p t id e , t h a t i s ,

a pre-cx f u n c t io n . I t i s c l e a r t h a t a more o r l e s s com plex

s e t o f c a sc a d e c o n t r o l s i s o p e r a t iv e in th e tem p o ra l e x p r e s s io n

o f HSV-induced p o ly p e p t id e s , p ro b a b ly a c t in g a t th e

t r a n s c r ip t io n a l l e v e l .

T reatm ent o f 1-JSV-infected c e l l s w ith p r o te a s e

- 31 -

i n h i b i t o r s f a i l e d to show ra p id p o s t - t r a n s l a t i o n a l c le a v a g e

o f v ir u s - in d u c e d p o ly p e p t id e s (H oness and Roizm an, 1973;

P e r e ir a e t a l . , 1 9 7 7 ) . In c o n t r a s t , ex p o su re o f v i r u s -

in f e c t e d c e l l s to th e i n h i b i t o r f o r lo n g e r p e r io d s o f tim e

showed a l t e r e d p r o c e s s in g o f s e v e r a l p o ly p e p t id e s (I).

M acDonald, M. Suh and U .S . M arsden, u n p u b lish e d d a t a ) .

A p p rox im ately 20 p h o sp h o r y la ted p o ly p e p t id e s have been

i d e n t i f i e d i n H S V -in fected c e l l s (M arsden e t a l . , 1978;

Bookout and L evy, 1980; W ilcox e t a l . , 1 9 8 0 ) . The

p h o sp h o r y la t io n pathway may d i f f e r betw een p o ly p e p t id e s and

th e l e v e l o f p h o sp h o r y la t io n may p la y a p a r t in th e b in d in g .

a f f i n i t y o f c e r t a in p h o sp h o p r o te in s f o r DNA (F enw ick e t a l . ,

1 9 80; W ilcox et, a l . , 1 9 8 0 ) . The p r o c e s s in g o f th e

p h o sp h o r y la te d c* p o ly p e p t id e Vmw IE 175 (IGP4) h a s been s tu d ie d

in some d e t a i l . T h is p o ly p e p t id e e x i s t s i n th r e e form s -

d i f f e r i n g in ap p aren t m o le c u la r w e ig h t , and i t i s th o u g h t

th a t ICP4a i 3 p a s s iv e ly tr a n sp o r te d in t o th e n u c le u s , where

i t i s p r o c e s se d in t o ICP4b th en ICP4c ( P e r e ir a e t a l . , 1977;

Fenw ick e t a l . , 1 9 7 8 ) . The n a tu r e o f t h e s e p r o c e s s in g e v e n ts

has n o t been d e term in ed , a lth o u g h a m utant w ith a l e s i o n in

th e ICP4 gen e h as been s tu d ie d w hich a ccu m u la tes ICP4a and

f a i l s to tr a n s p o r t i t in t o th e n u c le u s (C ou rtn ey and P o w e ll ,

1975; C ourtney e t a l . , 1976; C ab ral e t a l . , 1 9 8 0 ) . The

p h o sp h a te m o ie ty i s s t a b ly a s s o c ia t e d w ith ICP4b but c y c l e s

on and o f f ICPAa and ICP4c (W ilcox e t a l . , 1 9 8 0 ) . Bookout

and Levy (1 9 8 0 ) have r e c e n t ly d e s c r ib e d fo u r form s o f ICP4,

o n ly two o f w hich a re found in th e c y to p la sm .

A number o f nSV -induced p o ly p e p t id e s a re g ly c o s y la t e d ,

jjrob ab ly by h o s t c e l l enzym es (K e lle r e t a l . , 1 9 7 0 ) .

G ly c o s y la t io n o f p o ly p e p t id e s o c c u r s in s i t u , soon a f t e r

p o ly p e p t id e s y n t h e s is and t h e ir b in d in g to c e l l membranes

- 32 -

(S p ear and Roizm an, 1 9 7 0 ) . F iv e m ajor g ly c o p r o t e in s (gA, gB,

gC, gl) and gB) have been i d e n t i f i e d in th e v i r io n e n v e lo p e

and in f e c t e d c e l l membrane (S p ear e t a l . , 1970; S p ea r , 1976;

M arsden e t a l . , 1976; Baucke and S p ea r , 1 9 7 9 ) . gA i s r a r e in

v i r i o n s , and gB f u n c t io n s i n v ir u s p e n e t r a t io n and c e l l

f u s io n (M a n serv ig i and S p ea r , 1 9 77; S arm ien to e t a l . , 1 9 7 9 ) .

T hese two g ly c o p r o t e in s a re th o u g h t to o r ig in a t e from th e

same p r e c u r so r p o ly p e p t id e (E b e r le and C ou rtn ey , 1 9 8 0 b ) .

gC h a s an in h ib i t o r y e f f e c t upon c e l l f u s io n , and i s n o t

e s s e n t i a l f o r v ir io n i n f e c t i v i t y (M a n serv ig i and S p ea r , 1977;• .

Ruyechan e t a l . , 1 9 7 9 ) . The l e a s t s tu d ie d o f th e g ly c o p r o t e in s ,

gE, i s e s s e n t i a l f o r v i r io n i n f e c t i v i t y and a c t s a s an Fc

r e c e p to r f o r y ' -g lo b u l in , a f u n c t io n w hich may i n h i b i t th e

h o s t immume c y t o l y t i c r e sp o n se m ed ia ted by v i r a l g ly c o p r o t e in s

ex p o sed a t th e in f e c t e d c e l l s u r fa c e (Baucke and S p ea r , 1979;

P ara e t a l . , 1 9 80; M a ch tig er e t a l . # 1 9 8 0 ) .

G ly c o s y la t io n o f p r e c u r so r p o ly p e p t id e s i s a f t o r y

e v e n t o c c u r r in g in two s ta g e s * r a p id a d d it io n o f an

o lig o m a n n o sy l co r e fo llo w e d by s lo w e r a d d it io n o f fu c o s e and

s i a l i c a c id r e s id u e s (H oness and Roizm an, 1 9 7 5 b ;E isen b e rg

e t a l . , 1 9 7 9 ; Cohen e t a l . , 1 9 8 0 a ) . A ttem p ts have been made

to i d e n t i f y p r e c u r s o r form s by im m u n o lo g ica l a n a l y s i s , th e

u se o f d ru gs w hich b lo c k th e g ly c o s y la t io n pathw ay, tr e a tm e n t

o f p r o c e s se d p o ly p e p t id e s w ith g l y c o s id a s e s , and by p u ls e - c h a s ebetry

e x p e r im e n ts . C o n c lu s io n s h a v e ^ c r i t ic i s e d on th e grou n d s t h a t

breakdown p r o d u c ts and tr u e p r e c u r s o r s a r e d i f f i c u l t to

d i s t i n g u i s h . N e v e r t h e le s s , tw o -d im e n s io n a l e l e c t r o p h o r e t i c

a n a l y s i s s t r o n g ly i n d i c a t e s th a t 16 s p e c ie s a r e in v o lv e d in

th e fo r m a tio n o f gB, 1 5 -2 0 in gC, and 11 i n gD (Cohen e t a l . ,

1980a; Haarr and M arsden, 1 9 8 1 ) .

IISV-1 g ly c o p r o t e in s a re a l s o s u lp h a te d , and a su lp h a te d

- 33 -

breakdown p ro d u ct o f gl) i s e x c r e te d from th e i n f e c t e d c e l l

(R .G . Hope and H .S . M arsden, p e r s o n a l co m m u n ica tio n ).

The m ajor g ly c o p r o t e in o f P R V -in fec te d c e l l s i s su lp h a te d

su b seq u en t to g ly c o s y la t io n and th en e x c r e te d (E r ick so n and

K aplan, 1973; K aplan and B en -P o ra t, 1 9 76; P e n n in g to n and

McCrea, 1 9 7 7 ) /

Much o f th e p r e s e n t u n d e r sta n d in g o f H S V -sp e c if ie d

f u n c t io n s , and th e l o c a t io n o f e s s e n t i a l g e n e s i n th e genom e,

h as come from a n a ly s e s o f m u ta n ts . In t h i s r e s p e c t th e

commonest and m ost g e n e r a l ly u s e f u l ty p e i s th e t s m utant,

w hich was f i r s t d e s c r ib e d i n HSV by Subak-Sharpe (1 9 6 9 ) .

S in c e th en a la r g e number o f t s m u tan ts have been i s o l a t e d .

23 com p lem en ta tion grou p s in HSV-l and 20 i n HSV-2 w ere

i d e n t i f i e d in a c o l la b o r a t iv e s tu d y by S c h a f fe r e t a l . ( 1 9 7 8 ) ,

and th e r e c e n t u se o f s p e c i f i c m u ta g e n e s is o f DNA fra g m en ts

h as added a t l e a s t th r e e more to HSV-l (Chu e t a l . , 1979;

C onley e t a l . , 1 9 8 1 ) . L in e a r g e n e t ic l in k a g e maps f o r t s ,

p la q u e m orphology and drug r e s i s t a n c e m arkers have been

deduced (Brown e t a l . , 1973; S c h a f fe r e t a l . , 1 9 74;

Subak-Sharpe e t a l . , 1974; B en y esh -M eln ick e t a l . , 1974;

Timbury and C a ld er , 1976; Brown and Jam ieson , 1 9 7 7 ) .

R ecom b in ation in HSV was f i r s t n o ted by W ildy (1 9 5 5 ) ,

and in t e r t y p i c com p lem en ta tion and r ec o m b in a tio n by Timbury

and Subak-Sharpe ( 1 9 7 3 ) . R ep eated rounds o f m atin g and

r ec o m b in a tio n o c c u r d u rin g i n f e c t i o n , and reco m b in a n ts seem

to be produced by th e fo rm a tio n o f p a r t ia l h e t e r o z y g o te s

(Brown and R i t c h ie , 1975; R it c h ie e t a l . , 1 9 7 7 ) . E x c e l le n t

u se h as been made o f in t r a t y p ic (m arker r e s c u e ) and in t e r t y p ic

r ec o m b in a tio n to p h y s i c a l ly map IiSV m u ta tio n s and f u n c t io n s

on th e genome (W ilx ie e t a l . , 1977b; Marsden e t a l . , 1978;

Morse e t a l . , 1978; P r e s to n e t a l . , 1978; Stow and W ilk ie ,

1978; Stow e t a l . , 1978; H a ll ib u r to n , 1980; P a r r is e t a l . ,

1980; C hartrand e t a l . , 1981; S a n d r i-G o ld in e t a l . , 1 9 8 1 ) .

HSV-l/HSV-2 i n t e r t y p i c reco m b in a n ts have p roved e s p e c i a l l y

p o w erfu l in a s s ig n in g s p e c i f i c p o ly p e p t id e s and b io c h e m ic a l

f u n c t io n s to r e g io n s o f th e genom e, s in c e r e s t r i c t i o n

e n d o n u c lea se mapping a l lo w s c r o s s o v e r s to be a c c u r a t e ly

d e f in e d , and th e p o ly p e p t id e p r o f i l e s o f th e two s e r o ty p e s

are s u f f i c i e n t l y d i f f e r e n t to d i s t i n g u i s h th e o r ig in o f

many o f th e p o ly p e p t id e s in d u ced by r e c o m b in a n ts . A summary

o f th e mapping p o s i t i o n s o f s e v e r a l p o ly p e p t id e s i s shown

in F ig u r e A1.5*

Enzyme a c t i v i t i e s in d u ced by HSV

S e v e r a l en zym atic a c t i v i t i e s a re in d u ced i n H S V -in fected

c e l l s . T hese in c lu d e p y r im id in e d e o x y n u c le o s id e k in a s e (dPyK)

(K it and Dubbs, 1 9 6 3 a ) , BNA p o lym erase (K e ir and G old, 1963)>

a l k a l in e d e o x y r ib o n u c le a se (M orrison and K e ir , 1 9 6 8 ) ,

r ib o n u c le o t id e r e d u c ta se (Ponce de Leon e t a l . , 1977;

H uszar and B a c c h e t t i , 1 9 8 1 ) , a d e n y lic a c id sd e o x y th y m id in e

5 1-p h o s p h o tr a n s fe r a s e (Jam ieson e t a l . , 1976a; F a lk e e t a l . ,

1 9 8 1 ) , d e o x y r ib o p y r im id in e t r ip h o s p h a ta s e (W ohlrahb and

F ra n ck e , 1 9 8 0 ) , and a p r o t e in k in a s e (B lu e and S to b b s , 1 9 8 1 ) .

The in d u c t io n o f d e o x y c y t id in e d eam inase (Chan, 1977;

B. B u t ia , p e r s o n a l com m unication) and d e o x y c y t id y la t e

d eam in ase (H o lto n and K e ir , 1974; l a n g e l i e r e t a l « , 1 9 7 8 )

i s d i s p u t e d . A BNA g y r a se has been so u g h t but n o t y e t

d em on stra ted ( Francke and M a rg o lin , 1 9 8 1 ) .

The i s o l a t i o n o f an HSV-2 t s m utant w h ich a l s o h a s a

n o n - le t h a l m u ta tio n in th e s t r u c t u r a l gen e f o r th e a lk a l in e

d e o x y r ib o n u c le a se has d em on strated th a t t h i s enzyme i s a t

l e a s t in p a r t coded by th e v ir u s genome (F rancke e t a l . , 1 9 7 8 ) .

The p u r i f i e d enzyme h as a ra.wt. o f 9 0 ,0 0 0 , but i t s f u n c t io n

- 35 -

in v iv o i s a m a tter o f s p e c u la t io n ( S t r o b e l - F id le r and F rancke,

1 9 8 0 J . Moss, e t a l . (1 9 7 9 ) mapped th e n u c le a s e gen e to

0 .1 2 - 0 .2 1 f r a c t i o n a l genome u n i t s .

The v ir u s -c o d e d BNA p o lym erase h a s a m .w t.

o f 1 5 0 ,0 0 0 and seem s to be a c t i v e i n v i t r o a s a monomer

(P o w e ll and P u r ifo y , 1 9 7 7 ) . T h is enzyme i s r e a d i ly

d is t in g u is h e d from th e c e l l BNA p o ly m era se by im m u n o lo g ica l

(K e ir e t a l . , 1 9 6 6 ) , g e n e t i c ' (Aron e t a l . , 1 9 7 5 ) and i n h i b i t o r

s t u d i e s . The HSV BNA p o ly m era se i s v e r y s e n s i t i v e to

i n h i b i t i o n by p h o sp h o n o a c e t ic a c id (PAA; Mao and R obishaw ,

1 9 7 5 ; Hay and Sub ak -S h arp e, 1 9 76) and by a c y c lo g u a n o s in e

( a c y c l o v i r , ACV) in th e t r ip h o s p h a te form (Furman e t a l . ,

1 9 7 9 ; Barby e t a l . , 1 9 8 0 ) . M utant v ir u s e s r e s i s t a n t to PAA

p o s s e s s a P A A -r e s is ta n t . BNA p o ly m era se ( J o fr e e t a l . , 1977;

P u r ifo y and P o w e ll, 1 9 7 7 ) , s u g g e s t in g t h a t a s in g l e lo c u s i s

in v o lv e d i n P A A - s e n s i t iv i t y . A C V - s e n s it iv i t y , on th e o th e r

hand, i s a s s o c ia t e d w ith two d i s t i n c t l o c i c o r r e sp o n d in g to

th e BNA p o lym erase and dPyK (Crumpacker e t a l . , 1 9 8 0 ) . T h is

d i f f e r e n c e i s due to d i r e c t i n t e r a c t i o n o f PAA w ith th e

p o ly m e r a se , w hereas ACV i s f i r s t p h o sp h o r y la te d by dPyK, th e n c e

to th e tr ip h o s p h a te w hich i n h i b i t s th e p o ly m era se ( E lio n e t a l . >

1 9 7 7 ) . I t i s p o s s ib l e t h a t two c i s t r o n s a r e in v o lv e d i n

BNA p o ly m era se e x p r e s s io n , s in c e t s m u tan ts from two

co m p lem en ta tio n g ro u p s, w hich a re c l o s e l y a s s o c ia t e d i n th e

r e g io n o f 0 .4 f r a c t i o n a l genome u n i t s (C hartrand e t a l . ,

I 9 6 0 ) , both e x p r e s s a t s BNA p o lym erase (P u r ifo y and P o w e ll , 1 9 8 1 ) .

The v ir u s -c o d e d dPyK, a s i t s name s u g g e s t s , i s a b le to

p h o sp h o r y la te both th y m id in e and d e o x y c y t id in e , and seem s a l s o

to e x h ib i t a n u c le o s id e p h o sp h o tr a n s fe r a s e a c t i v i t y (Jam ieson

e t a l . , 1974 and 1 9 7 6 a ;J a m ieso n and Subak -S h arp e, 1 9 7 4 ) .

S tro n g e v id e n c e th a t t h i s enzyme i s v ir u s -c o d e d com es from

- 56 -

im m u n o lo g ic a l, b io c h e m ic a l and g e n e t ic s t u d ie s (K lem perer

e t a l . , 1967; Buchan and W atson, 1 9 6 9 ; Buchan e t a l . , 1970;

T h o u le s s , 1 972; Aron e t a l . , 1 9 7 3 ) . The a c t i v e p o ly p e p t id e

in d u ced i n v iv o o r produced in v i t r o by t r a n s la t io n o f mRNA

h as an ap p aren t m .w t. o f 4 0 ,0 0 0 (H oness and W atson, 1974;

Summers e t a l . , 1975; T h o u le ss and W ildy, 1 9 75; P r e s to n , 1977;

Cremer e t a l . , 1977 and 1 9 7 8 ) . T w o -d im en sio n a l g e l

e le c t r o p h o r e s i s o f H SV-induced p o ly p e p t id e s showed th a t t h i s

p o ly p e p t id e may e x i s t i n th r e e d i f f e r e n t l y ch arged form s

(H aarr and M arsden, 1 9 8 1 ) . P r e s to n and McGeoch (1 9 8 1 )

i d e n t i f i e d a s l i g h t l y s m a lle r p o ly p e p t id e e x p r e s se d in v i t r o

and in v i v o , c o n c lu d in g t h a t t h i s was th e r e s u l t o f f a l s e

i n i t i a t i o n o f t r a n s l a t io n from a s i n g l e mRNA s p e c i e s .

The r e s u l t s o f ex p er im e n ts u s in g mixed i n f e c t i o n s o f dPyK

and dPyK v ir u s e s s u g g e s t t h a t th e a c t i v e enzyme i s

p ro b a b ly a rau ltim er (Jam ieson and S u b ak -S h arp e, 1 9 7 8 ) .

A number o f n u c le o s id e a n a lo g u e s i n h i b i t v i r u s grow th b ecau se

t h e y a r e p h o sp h o r y la ted by dPyK, and th e n c e b ind to th e

BNA p o ly m era se o r become l e t h a i l y in c o r p o r a te d in t o n ew ly

s y n t h e s is e d v ir u s BNA. Among t h e s e a re ACV, b ro m o d eo x y cy tid in e ,

b rom od eoxyu rid in e and 1 -^ - a r a b in o s y l th ym in e (K it and Bubbs,

1 9 6 3 ^ ;C ooper, 1973; S c h ild k r a u t e t a l . , 1 9 75; M il le r e t a l . ,

1 9 7 7 ; E lio n e t a l . , 1 9 7 7 ; M u ller e t a l . , 1 9 7 9 b ) . Such

i n h i b i t o r s have been u sed to s e l e c t v ia b le dPyK~ m u ta n ts .

A lth o u g h dPyK a c t i v i t y i s n o t r e q u ir e d f o r v ir u s r e p l i c a t io n

in a c t i v e l y d iv id in g c e l l s , i t i s an e s s e n t i a l f u n c t io n f o r

grow th in r e s t i n g c e l l s , f o r f u l l n e u r o v ir u le n c e , and f o r

norm al e s ta b lis h m e n t o f la t e n c y (Jam ieson e t a l . , 1974;

F ie ld and B arby, 1978 and 1980; Barby e t a l . , 1 9 8 1 ) .

The c o n s tr u c t io n o f b a c t e r ia l p la sm id s c o n ta in in g th e

- 37 -

HSV-1 dPyK gen e (HSV-1 Bamil l £ mapping a t a p p ro x im a te ly 0 .3

f r a c t i o n a l genome u n i t s ) has opened th e way to u n d ersta n d in g

th e m o le c u la r g e n e t i c s o f t h i s f u n c t io n (W ilk ie e t a l . , 1979a;

B n q u ist e t a l . , 1979; C o lb ere-G a ra p in e t a l . , 1 9 7 9 ) . The

DMA seq u en ce o f th e gene has been d eterm in ed (M cKhight, 1 9 80;

Wagner e t a l . , 1981; P. McGeoch, p e r s o n a l co m m u n ica tio n ), and

v ia b le dPyK"" d e l e t io n m u tan ts have been c o n s tr u c te d in v i t r o

(W ilk ie e V a l . , 1980; S m ile y , 1 9 8 0 ) . The dPyK i s a ^

p o ly p e p t id e w h ich r e q u ir e s f o r i t s p r o d u c t io n i n th e in f e c t e d

c .e l l an oL f u n c t io n w hich a c t s a t th e t r a n s c r ip t io n a l l e v e l

(G a r f in k le and M cAuslan, 1974; Leung e t a l * , 1 9 8 0 ) . The

gene i s t r a n s c r ib e d from i t s own prom oter le f tw a r d s w ith

r e s p e c t to th e P o r ie n t a t io n o f th e genom e, and th e mRNA i s

n o t s p l i c e d (W ilk ie e t a l . , 1980; S m iley e t a l . , 1 9 8 0 ) .

The HSV-1 dPyK gen e has been u sed a s a s e p a r a te e n t i t y

from th e genome from w hich i t was d e r iv e d . C e l l

l i n e s la c k in g th e c e l l th y m id in e k in a s e have been b io c h e m ic a lly

tra n sfo rm ed u s in g in a c t iv a t e d HSV or DNA fra g m e n ts (Munyon

e t a l • , 1971; Jam ieson e t a l . , 1 9 7 8 b ;M a itla n d and M cD ougall,

19 77; W ig ler e t a l . , 1977)* U sin g a c lo n e d DNA fragm en t

c o n ta in in g th e dPyK g e n e , P e l l i c e r e t a l . (1 9 7 8 ) have r e p o r te d

t h a t th e v ir u s DNA i s in t e g r a t e d i n t o th e c e l l DNA, a lth o u ^ i

n o t .at a s p e c i f i c s i t e . O ther exogen ou s DNA seq u e n c es have

been s t a b ly in tr o d u c e d in t o c e l l s by em p loy in g th e dPyK gene

a s a s e l e c t a b l e m arker, i n some e x p e r im e n ts o o v a le n t ly l in k e d

to th e ex o g en o u s DNA (M antei e t a l . , 1979; W ig ler e t a l . , 1979)

A p p rox im ately 20 H S V -l-in d u ced DNA b in d in g p r o t e in s

have been i d e n t i f i e d , in c lu d in g the. oi p o ly p e p t id e s , th e

DNA p o ly m er a se , and p ro b a b ly th e a lk a l in e d e o x y r ib o n u c le a se

and dPyK ( B a y l i s s e t a l . , 1975; P o w e ll and P u r ifo y , 1976;

B ookout and L evy , 1980; Becker e t a l . , 1 9 8 0 ; Hay and Hay, 1 9 8 0 )*

Figure Al.lUPPER PANEL S tr u c tu r a l arrangem ent o f th e HSV-1

genom e. The genome co m p r ises two c o v a le n t ly l in k e d segm ents

(L, S ) , each o f w hich c o n s i s t s o f a u n iq u e seq u en ce (UL, Ug)

bounded by in v e r te d r e p e t i t i o n s (TH^, IR^, TRg, IR § ). '

and Us a re sh o r te n e d in t h i s diagram to em p h asise th e

l o c a t i o n s and o r i e n t a t io n s (arrow ed) o f th e a s e q u e n c e s , or

t e r m in a l r e p e t i t i o n s ( t r ) a t th e genome te r m in i and L-S

j o i n t . A lso shown a re th e b and c seq u e n c e s ( TRj, = b + a ,

I R1 * y TRg» c + a , IRg** c f+ a f ) . S i z e s a r e g iv e n in kbp.

A S in g le DNA m o le c u le c o n ta in s each segm ent i n e i t h e r

o r ie n t a t io n w ith e q u a l p r o b a b i l i t y , a s shown by th e la r g e r

a rro w s, a l lo w in g fo u r p erm u ta tio n s o f seq u en ce arran gem en t.

The genome s t r u c t u r e o f HSV-2 i s e s s e n t i a l l y i d e n t i c a l .

LOWER PANEL G en era tio n o f subm olar r e s t r i c t i o n

e n d o n u c le a se fra g m en ts from HSV DNA. A h y p o th e t ic a l

e n d o n u c le a se c le a v e s th e DNA in th r e e l o c a t i o n s . As a

co n seq u en c e o f in v e r s io n o f th e two seg m en ts, fo u r q u a r te r -

m olar fra g m en ts sp a n n in g th e L-S ju n c t io n (a -t -e , c + e ,

a-t-b , c + b ) , fo u r h a lf -m o la r fra g m en ts from th e te r m in i

(a , b, c , e ) , and one m olar fragm en t (d ) a re g e n e r a te d .

E x te n s io n o f t h i s argum ent shows t h a t i f one s e t o f

r e p e a t s a l s o c o n t a in s a r e s t r i c t i o n s i t e , h a lf -m o la r but

n o t q u a r te r -m o la r fra g m en ts would be g e n e r a te d . I f both

s e t s o f r e p e a t s a l s o c o n ta in e d a r e s t r i c t i o n s i t e , o n ly

m olar fr a g m en ts w ould r e s u l t .

In t h i s F ig u re th e fo u r arran gem en ts have been

term ed P , 1^, I g , Igq, in accord an ce w ith th e n om en cla tu re

p ro p o sed by Roizman e t a l . (1 9 7 9 ) . R e s t r ic t io n maps o f

HSV BNA a re c o n v e n t io n a l ly d isp la y e d in th e P arran gem en t.

L II

IRl [iRs10.5 • 6.0

s120 25

TRl TRS10.5 6.0

t r t r t r0.4

Ul0.4 0.4

V Y Us# YI

a+e

a+b

SL

Figure A1.2 ( o v e r le a f )

S tr u c tu r e s o f h e r p e s v ir u s genom es and g e n e r a l

m odel f o r in te r c o n v e r s io n o f l i n e a r and c i r c u l a r fo r m s .

L in e a r genome s t r u c t u r e s o f th e fo u r main

groups ( I - I V ) a re shown to s c a le o n th e r ig h t hand p a g e .

Two sub grou p s have been t e n t a t i v e l y in c lu d e d in group I .

In each c a s e th e m .w t. (x 10~^) and o b serv ed o r ie n t a t io n s ( f i l l e d

arrow heads) o f and, where a p p r o p r ia te , Ug, a re in d ic a t e d .

R epeated se q u e n c e s a re shown a s r e c t a n g le s , and t h e ir

r e l a t i v e o r i e n t a t io n s by arrow s and by u se o f th e l e t t e r s

a , b, c and t h e i r com plem ent a 1, b 1, c 1 ( —A a n d a^ , bn

s i g n i f y m u lt ip le tandem r e p e a t s ) . The u se o f l e t t e r s d o es

n o t d en o te s i m i l a r i t y o f seq u en ce o r f u n c t io n in th e s e

r e g io n s betw een d i f f e r e n t genom es.

I t i s known th a t th e genom es i l l u s t r a t e d are

t e r m in a lly red u n d a n t, a lth o u g h th o s e o f PRV and EHV-1 have

n o t y e t been d i r e c t l y shown to be s o . N e v e r t h e le s s , t h i s

f e a tu r e makes i t p o s s i b l e , a t l e a s t in p r i n c i p l e , to

c i r c u l a r i s e h e r p e s v ir u s genom es by l i g a t i o n su b seq u en t to

th e a d t io n o f an e x o n u c le a s e . The fo u r g rou p s o f p u ta t iv e

c ir c u la r form s a re shown on th e l e f t hand p a g e , n o t drawn

to s c a l e . O r ie n ta t io n s o f r e p e a te d se q u e n c es ( r e c t a n g le s )

have been o m itte d f o r c l a r i t y . P o s i t i o n s o f l i g a t i o n on

o p p o s ite s tr a n d s o f th e d u p lex a r e in d ic a t e d ( ▼ ) .

C le a r ly , th e r e v e r s e p ro ce ss ., s in g l e s tra n d ed c le a v a g e on

each s id e o f th e te r m in a l r e p e a t w ith in a c i r c u l a r DNA

m o le c u le fo llo w e d by r e p a ir o f th e s in g l e s tra n d ed

te r m in i, would g e n e r a te a l i n e a r genom e. C lea v a g e may

o ccu r a t s e v e r a l p o s s ib l e l o c a t io n s w ith in th e tandem

r e i t e r a t i o n s o f grou p s I I and I I I , g e n e r a t in g th e

h e te r o g e n e o u s te r m in i o b serv ed in v ir io n DNA.

6 8 * * 9 *D - .<■ »■ - ■CEKK3 HSVa b b'a'c' ca

« 6 6 »«■ 8 *

D <->------------QU*HU BMVab ba'c' ca«■ 115 22 >□ ----------------------------------- «— »------------------------------------ c n « » - qa b B’a’c' ca

HCMV

lb

II

IIIan

I V

66 6— • — — i u * n p r v

a' b' b a 74 4" 8

— ■ 0 * 0 EHV-1a' b' b a

M 87 —i ------------------ » nnnnnw i ebv ,h v pa n b n a n

-V> 70»------------ j I HVS,HVA

62 ____I— I------------ *------------[ZD CCVa b a b

Figure A1.3

Mean GC c o n t e n t s o f h e r p e s v ir u s DNAs, tak en

from H oness and Watson (1 9 7 7 b ) . F i l l e d c i r c l e s in d ic a t e

human h e r p e s v ir u s e s .

A b b r e v ia t io n s o f v ir u s n om en cla tu re o th e r th an

th e o b v io u s a re a s f o l l o w s .

SpM - s p id e r monkey h e r p e s v ir u s(c e b id h e r p e s v ir u s 3)

IBR - i n f e c t i o u s b o v in e r h i n o t r a c h e i t i sv ir u s

SqM - s q u ir r e l monkey h e r p e s v ir u s(h e r p e s v ir u s ta m a rin u s)

GCMV - g u in e a p ig c y to m e g a lo v ir u s

MaCMV - marmoset c y to m e g a lo v ir u s

SA6 - s im ia n c y to m e g a lo v ir u s

HhCMV - r h e su s c y to m e g a lo v ir u s

VeCMY - v e r v e t c y to m e g a lo v ir u s

FelLT - f e l i n e i n f e c t i o u s la r y n g o t r a c h e i t i sv ir u s

AvILT - a v ia n i n f e c t i o u s la r y n g o t r a c h e i t i s

oO SpM

O IBR

O B-virus

PRV

8

• HSV-2HSV-I

SqM EHV-3

Tree shrewBMV

OOoo•

8

# EBV(HR-I)• EBV( B95-8) O MCMV

• HCMV8 GCMV EHV-2 « EHV-I

0° c c vFrog virus 4

MaCMV SA6 RhCMV VeCMV

u Trout Rat HVT MDV

FelLT VZV HVS LHV

AvILT

Oooo

O rabbi t O dog L 1, J.

3Q 40 50 60

% GC

70 80

Figure A1.4 (overleaf)

Immediate e a r ly (IE) t r a n s c r ip t io n a l program o f

HSV-1. The genome i s shown to s c a le in the c o n v e n tio n a l

manner, e x ce p t th a t two r e g io n s in have been om itted

f o r c l a r i t y ( A r ) , The s c a le a t the top o f th e F igure

shows c o o r d in a te s o f f r a c t io n a l genome le n g th .

The lo c a t io n s and d ir e c t io n s o f t r a n s c r ip t io n

o f IE mRNAs 1 -5 are shown (C lem ents e t a l . , 1977; Jones

e t a l . , 1977; C lem ents e t a l . , 1979; Anderson e t a l* , 1980a;

F .J . R ixon , p e r so n a l com m unication). A lso in c lu d e d are

s im ila r d a ta fo r th e 5 .0 kb mRNA cod ing fo r Vmw IE 136*(143)

(Anderson e t a l . , 1981; J . McLauchlan and J .B . C lem ents,

p e r so n a l com m unication). The p o s i t io n o f the in tr o n

common to the gen es fo r IE mRNAs 4 and 5 i s in d ic a te d

(Watson e t a l . , 1981; F .J . R ixon, p erso n a l com m unication).

I t i s su sp ec ted th a t th e gene fo r IE mRNA 1 a ls o c o n ta in s

an in tr o n , because th e mRNA i s c o n s id e r a b ly sm a ller than

th e r e g io n o f ENA encoding i t , whereas th e gen es fo r

IE mRNAs 2 and 3 c o n ta in no d e te c ta b le in tr o n s (F .J . R ixon,

p e r so n a l com m unication). The r e g io n s in the ENA encoding

th e 5 1 term in i o f IE mRNAs 1 , 3 , 4 and 5 have been lo c a te d

(Mackern and Roizman, 1980) and n u c le o t id e seq u en ces

determ ined fo r th e se r e g io n s o f IE mRNAs 3 , 4 and 5

(M-J. M urchie, p erso n a l com m unication). S im ila r ly th e

r e g io n s encod in g the 3' term in i o f IE mRNAs 1 , 2 and 5 are

known (S e c t io n 3 o f R e su lts ; M-J. Murchie and F .J . R ixon,

p e r so n a l com m u nication s). The sequence cod in g f o r th e

tr a n s la te d r eg io n o f IE mRNA 5, which l i e s co m p lete ly

w ith in Ug, has been determ ined (M-J. M urchie, p erso n a l

com m unication). The 5 .0 kb mRNA cod in g fo r Vmw IE 1 3 6 '(1 4 3 )

i s ex p ressed m in im ally under IE c o n d it io n s and th e r e fo r e

th ere i s some h e s i t a t io n in i t s in c lu s io n in the IE

c l a s s . A 1 .2 Jcb mRNA 3 1 -c o te r m in a l w ith th e 5*0 kb mRNA

encodes a d i f f e r e n t p o ly p e p tid e , th e co d in g r e g io n fo r

which l i e s o u ts id e th a t fo r Vmw IE 136* (143 )

(J . M clauchlan and J .B . C lem ents, p e r so n a l com m unication).

Below each mRNA i s in d ic a te d th e s i z e in kb o f the

p o ly a d en y la ted mRNA determ ined by m ethylm ercurie hydroxide

agarose g e l e le c tr o p h o r e s is (Watson e t a l . , 1979;

T alley-B row n and M i l l e t t e , 1979; Anderson e t a l . , 1980a and b ) .

In v i t r o t r a n s la t io n o f i s o la t e d mRNAs perform ed in

the s tu d ie s m entioned above, and th e mapping o f

p o ly p e p tid e s u t i l i s i n g HSV-l/HSV-2 in t e r t y p ic recom binants

(P resto n e t a l . , 1978; Marsden e t a l . , 1978; Morse e t a l . ,

1 9 7 8 ), have a llo w ed i d e n t i f i c a t io n o f the p o ly p e p tid e

encoded by each IE mRNA. At th e f o o t o f th e F igure th e

e q u iv a le n t p o ly p e p tid e s in the nom enclature o f H oness

and Roizman (1973 and 1974) are g iv e n .

CO CM(O CM

" ICO N-

cm (D CM

i

Figure A1.5

Map lo c a t io n s o f HSV-induced p o ly p e p tid e s

(Marsden e t a l , , 1978; P resto n e t a l . , 1978; H .S . Marsden,

p e r so n a l com m u nication ).

Each number (X) in d ic a t e s the apparent m.wt.

(x 10"^ ); f u l l nom enclature i s V X o r , in th e c a se o f

im m ediate e a r ly (IE ) p o ly p e p t id e s , Vmw IE X. P o ly p e p tid e s

w ith th e s u b s c r ip t "1" have no r e a d ily r e c o g n isa b le type 2

e q u iv a le n t , and th o se w ith th e su b s c r ip t 112 ” have no

r e a d ily r e c o g n is a b le type 1 e q u iv a le n t . Other p o ly p e p tid e s

are in d ic a te d by the m .wt. o f the HSV-1 form and have, no

s u b s c r ip t . A ccurate lo c a t io n s o f IE p o ly p e p tid e s from

t r a n s c r ip t mapping d a ta are shown in F igu re A 1 .4 .

immediateearlypolypeptides

methionine-

labelled

polypeptides

phosphorylated

polypeptides

glycosylated

polypeptides

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0

273 136’(143)

136T130) 103

90o 65,6467i 43

38,37.8

136’(143) 66’(66)

65’(65)<i

58 381

45-j 36o

67-8532,34,35

P A l l T B

M A T E R I A L S

A N D

M E T H O D S

i

- 38 -

MATERIALS

Vir u s e s

HSV-1 Glasgow s t r a in 17 syn* was i s o la t e d in Glasgow by' *4* ^])r C .A .P. R oss, and m utants syn tsB and syn tsD were in d ir e c t ly

•, - ^ - - - 4,

d er iv ed therefrom (Brown et; a l . , 1 9 7 3 ) .

HSV-1 s t r a in USA-8 syn* was i s o la t e d in P h ila d e lp h ia from

the l e f t tr ig e m in a l g a n g lio n o f a fem ale who d ied from

lym ph ocytic lymphoma (L onsdale e t a l . , 1 9 7 9 ) .

HSV-2 s t r a in HG52 was i s o la t e d from an a n a l l e s io n o f a

fem ale p a t ie n t in Edinburgh, and m utants t s l and t s 6 were

d er iv ed from t h i s s t r a in (Timbury, 1 9 7 1 ) .

liSV-l/HSV-2 in t e r t y p ic recom binant v ir u s e s are d e sc r ib ed

in F igure C 4 .3 .

The p seu d o ra b ies v ir u s s t r a in used was th a t o r ig in a l ly

i s o la t e d by Dr. A .S . Kaplan (Kaplan and V a tte r , 1 9 5 9 ) .

Ce l l s

The con tin u ou s c e l l l in e used was baby ham ster k idney

c lo n e 13 (BHK C13; MacPherson and S to k er , 1 9 6 2 ) .

B a c te r ia

E sc h e r ic h ia c o l i K12 s t r a in HB101 was used as h o s t

to p lasm id s (Boyer and R o iillan d -D u sso ix , 1 9 6 9 ) .

E sc h e r ic h ia c o l i met~"r~~m~" ( cI q^ S 7) used f o r the

growth o f b acter iop h age lambda was g iv en by Dr D. R i t c h ie .

B a c te r ia fo r the p rod u ction o f r e s t r i c t i o n en d o n u clea ses

are d e sc r ib e d in Table B l . l .

T issu e c u ltu r e media and s o lu t io n s

C e l ls were grown in th e Glasgow m o d if ic a t io n o f E a g le ’ s

medium (Busby e t a l . , 1964) supplem ented w ith 100 u n it s /m l

- 39 -p e n i c i l l i n , 1 0 0 /Mg/ml strep to m y cin , 0 . 2 yMg/ml a n tim y co tic

a g en t n -b u ty l-p -h y d ro x y b en zo a te , and 0 . 0 0 2 $ w t /v o l phenol r e d .

C a lf serum and p oo led human serum were prepared in the

I n s t i t u t e o f V ir o lo g y .

The fo l lo w in g com p osite s o lu t io n s were u sed .

E a g le ’ s medium c o n ta in in g x $ v o l /v o l c a l f serum;* _

ECx c o n ta in in g 10$ v o l /v o l D ifco tr y p to se phosphate

broth (E P );

E a g le 's medium c o n ta in in g s.% v o l /v o l human serum;

E a g le ’ s medium la c k in g orth oph osp hate c o n ta in in g

1 $ v o l /v o l c a l f serum.

P hosphate b u ffered s a l in e (PBS) co n ta in ed 0 .1 7 M NaCl, 0*0034

M KC1, 0 .0 0 1 M Na^HPO ., 0 .0 0 2 M KH2 PO4 in d i s t i l l e d w ater,

pH7• 2 .

PBSCa was PBS c o n ta in in g 5$ v o l /v o l c a l f serum.4 ^

V ersene c o n s is te d o f 0 .0 0 6M EDTA in PBS c o n ta in in g 0 .0 0 1 5 $m 4

w t/v o l phenol r e d .

T r is s a l in e was 0 .1 4 M NaCl, 0 .0 3 M KC1, 0 .0 0 0 2 8 M NaH2 ^ 4 >

1 mg/ml d e x tr o s e , 0 .0 0 1 5 $ w t /v o l p h en ol red , 0 .0 2 5 M T ris-H C l,r

pH 7 .4 supplem ented w ith 100 u n its /m l p e n i c i l l i n and 100 /<g/m l

strep to m y cin .

T ryp sin -versen e com prised one volume o f 0 .2 5 $ w t /v o l D ifco* ... *

tr y p s in in T r is s a l in e p lu s fou r volum es o f v e r se n e .

Giemsa s t a in c o n s is te d o f 1 .5 $ v o l /v o l su sp en sion o f Giemsa

in g ly c e r o l , heated to 5 6 ° fo r 90-120 min and d ilu te d w ith

an equal volume o f m ethanol.

R ad iochem icals

The fo l lo w in g r a d io is o to p e s were o b ta in ed from The

R a d io c h e m ic a l C e n t r e , Amersham, B u ck s .

ECx

ETCx

EHux

EC l-Pi

- 4 0 -

d eo x y n u c leo sid e (<*->2P) tr ip h o sp h a te s (dATP, PB164; dCTP, PB165

dGTP, PB166; dTTP, PB167; each a t 3 1 4 .8 TBq/mmol);

ad en osin e ( y - 52P) tr ip h o sp h a te (PB10218; > 185 TBq/mmol);

^2P orthophosphate (PBS11).

C hem icals

The f o l lo w in g firm s su p p lied th e l i s t e d c h e m ic a ls .

Agarose (ty p e 1 ) , Trizma b a se , eth id ium brom ide, 5$ v o l /v o l

s t e r i l e bovine serum album in, p o ly v in y lp y r o ll id o n e , bovine

serum albumin (RIA g r a d e ), r ib o n u c le a s e s A and Tl f y e a s t

RNA and lysozym e - Sigma (London) Chem ical Company, K in gston -

upon-Thames, London.

N ,N ,N ’ ,N 1-te tr a m e th y le th y le n e d ia m in e (TEMED), h y d r o x y la p a t ite ,

ammonium p ersu lp h a te and B iorex 70 (100-200 mesh) - B iorad

L a b o r a to r ie s , Richmond, C a l i fo r n ia .

Dextran su lp h a te , F i c o l l 400 and Sephadex G50 P ine -

Pharm acia F ine C hem icals, U ppsala, Sweden.

U ltr o g e l AcA 34 - LKB, Bromma, Sweden.

D iam inoethyl c e l lu l o s e (DE52) -Whatman L td ., M aidstone, Kent.

B r a in -h e a r t in fu s io n - Oxoid L td ., B a s in g sto k e , H ants.

2 , 5 -d ip h e n y lo x a zo le (PPO), l - 4 - d i - ( 2 - ( 5 - p h e n y lo x a z o ly l ) )

benzene (POPOP), t r ic h lo r o a c e t ic a c id and b o r ic a c id -

K och-Light L a b o ra to r ie s , Colnbrook, B erk s.

D im ethyl su lp h a te (DMS) - A ld r ich Chem ical Co. L td .,

G illin gh am , D o rse t.

- 41 -

H ydrazine - Eastman-Kodak, R och ester , New York.

P ip e r id in e - Pluorochem L td ., G lossop , D erb y sh ire .

M ithramycin (" M ith racin ") - P f iz e r L td ., Sandwich, Kent.

A m p ic illin - Beecham R esearch L a b o r a to r ie s , B ren tfo rd ,

M id d lesex .

O ther c h e m ic a ls , where n ot s t a t e d , were o b ta in ed where

p o s s ib le in Analar grade from BDH C hem icals L td ., P o o le , D o r se t .

jdna

C a lf thymus DNA and salmon sperm DNA were o b ta in ed from

Sigma L td ., and 0X i?4 RP I DNA from N ew England B io la b s ,

B e v er ly , M a ssa c h u sse tts .

P r o fe s so r D. S h e r r a tt k in d ly prov id ed pAT153 DNA.

Human cy to m eg a lo v iru s DNA ( s t r a in AD169) was g iv en by

Dr M. V/e s t s tr a t e .

DNA was prepared and g e n e ro u sly su p p lie d by Dr J . M.

W halley from a p la q u e -p u r if ie d A u str a lia n i s o l a t e (HVS 25)

o f equid h e r p e sv ir u s type I ob ta in ed from Dr II. Sabine o f

th e Department o f V eter in a ry M ed icin e, U n iv e r s ity o f Sydney.

Enzymes

R e s t r ic t io n en d o n u c lea ses n o t prepared from b a c te r ia

in t h i s s tu d y , and o th e r enzymes a c t in g upon DNA (T4 DNA

l i g a s e , b a c t e r ia l a lk a l in e p h osp h atase , E, c o l i DNA

polym erase I and th e Klenow fragm ent th e r e o f ) were purchased

from New England B io la b s , or from B ethesda R esearch

L a b o r a to r ie s , R o c k v il le , Maryland, or from th e B oehringer

C orporation (London) L td ., Lewes, E ast S u sse x .

D eoxyrib on u clease I was ob ta in ed from the W orthington

B ioch em ica l C orp oration , F reeh old , New J e r s e y .

- 42 -

T4 p o ly n u c le o t id e k in a se was su p p lie d by P-L /

B io ch em ica ls I n c . , M ilwaukee, Y /isconsin .

R e s t r ic t io n en d on u cleases Xhol and Smal were th e

generou s g i f t s o f Dr J . Arrand, and S s t I o f Dr P . R igby.

Other m a te r ia ls

P e t r i d is h e s - Plow la b o r a t o r ie s , I r v in e , S c o tla n d .

K odirex X -ray f i lm , X-Omat H X -ray f i lm and 1X80

d e v e lo p er - Kodak L td ., London.

Amfix - May and Baker L td ., Dagenham.

T eflo n tape - th e M innesota M ining and M anufacturing

Company, USA.

N it r o c e l lu lo s e membrane f i l t e r in s h e e t (BA85) and

grid d ed d is c form s - S c h le ic h e r and S c h i i l l , D a sse l, West Germany.

Commonly used s o lu t io n s and b u ffe r s

SSC 0 .1 5 M NaCl, 0 .0 1 5 M sodium o i t r a t e , pH 7 -5 ;

NTE 0 .0 1 M T ris-H C l, 0 .1 M NaCl, 0 .0 0 1 M EDTA,

pH 7 . 4 }

s c i n t i l l a t i o n 5 g PPO and 0 .3 g POPOP per l i t r e o f to lu e n e , f l u i d

METHODS

Growth o f c e l l s

BHK c e l l s were grown to co n flu e n c e in 80 oz r o l l e r

b o t t l e s a t 37° i a 200 ml ETC10 under 5$ 002 in a i r . C e l ls*

were h a rv ested by w ashing tw ic e w ith tr y p s in -v e r s e n e and

reseed ed a t 3 x 10^ c e l l s / r o l l e r b o t t le in ETC10. 50 mm- 6

d iam eter p l a s t i c P e t r i d is h e s were seeded a t 2 x 10

c e l l s / d i s h in 4 ml ETC10, th e m onolayers a t t a in in g c o n flu en ce

a f t e r 1 6 -2 4 t o a t 37°•

P rod u ction o f v ir u s s to c k s

80 oz r o l l e r b o t t le s c o n ta in in g a lm ost c o n f lu e n t BHK

c e l l s were in f e c t e d w ith v ir u s a t a m .o . i . o f 1 p . f . u . /

300 c e l l s . V irus was added in 40 ml EC5 per b o t t le and

in cu b a ted a t 3 1 ° . A fte r 2 -4 days in f e c t e d c e l l s were

3haken in to the medium, p e l le t e d a t 2000 r .p .ra . f o r 10 min

in an MSB C oo lsp in c e n tr i fu g e , and resuspended in 5 ml

PBSCa. The c e l l s were ruptured U3ing a C ole-P alm er

u lt r a s o n ic bath and c e n tr ifu g e d a t 2000 r .p .m . fo r 10 min

to remove c e l lu l a r d e b r is . The su p ern atan t was s to r e d in

s t e r i l e v i a l s a t - 7 0 ° . S t e r i l i t y ch eck s were c a r r ie d out

u s in g blood agar p la t e s and tr y p to se phosphate b ro th .

V irus t i t r a t i o n

V irus was d i lu t e d s e r i a l l y a t te n fo ld d i lu t io n s in

PBSCa. Drained BHK c e l l m onolayers i n 50 mm P e t r i d is h e s

were in o c u la te d w ith 0 .1 ml o f v ir u s d i lu t io n . V irus was

absorbed f o r 40 min a t th e tem perature o f subsequent

in c u b a tio n (3 1 ° or 3 7 °) and th en 4 ml EHu5 was added.

A fte r in c u b a tio n f o r 2 -3 days th e m onolayers were f ix e d and

s ta in e d w ith Giemsa s t a in f o r 15 min a t room tem p erature,

and p la q u es counted u s in g a d i s s e c t in g m icroscop e .

- 44 -

P laque p u r i f i c a t io n o f HSY

The medium (EHu5) was removed from P e tr i d is h e s

c o n ta in in g w e ll- s e p a r a te d in d iv id u a l p la q u es and th e

m onolayers were washed tw ic e w ith PBSCa. I n fe c te d c e l l s

form ing a s in g le p laque were taken up in to a f i n e l y drawn

P a steu r p ip e t t e and tr a n s fe r r e d to 0*5 ml PBSCa in a

s t e r i l e v i a l . Removal o f th e p laque from th e d is h was

c a r r ie d o u t under the dark f i e l d i l lu m in a t io n o f a Wild

M7A s te r e o s c o p ic d i s s e c t in g m icro sco p e . The v i a l was

so n ic a te d in an u lt r a s o n ic bath and r e le a s e d v ir u s

in o c u la te d in t e n fo ld d i lu t io n s onto BHK m onolayers in

P e t r i d is h e s , and p la q u es a llo w ed to d ev e lo p under 4 ml

EHu5. V irus was p laq ue p u r if ie d tw ic e more in th e same

way, and th e progeny from th e f i n a l p laque were used to

i n f e c t a s in g le 50 mm P e tr i d ish m onolayer. V irus from the

in f e c t e d m onolayer in cu b ated under 4 ml EC5 was t i t r a t e d

under a p p ro p r ia te c o n d it io n s and in some c a s e s used to

i n f e c t 80 oz r o l l e r b o t t l e s f o r p ro d u ctio n o f working

s to c k s o f v i r u s .

G en eration o f HSV-l/HSV-2 in t e r t y p ic recom binant v ir u s e s

The marker r e sc u e method o f Stow and W ilk ie (1976) was

u sed , in v o lv in g p r e c ip i t a t io n o f th e i n f e c t in g DNA a s a

ca lc iu m phosphate complex and osm otic sh ock in g o f th e c e l l s

by treatm en t w ith 25# d im eth y lsu lp h o x id e (DMSO) to s t im u la te

uptake o f th e DNA.1

I n ta c t DNA from a te m p e r a tu r e -s e n s it iv e m utant o f

HSV was m ixed ’a t 0 .4yu g /m l w ith 10 /Vg/ml c a l f thymus c a r r ie r

DNA and w ild type DNA r e s t r i c t i o n fragm ents i n H epes-

b u ffered s a l in e (HeBS: 8 .0 g /1 NaCl, 0 .3 7 g /1 KC1, 0 .1 g /1

Na2 HP04 , 1 .0 g /1 I - g lu c o s e , 5 .0 g /1 N -2 -h y d ro x y eth y lp ip era z in e

- 45 -N1-2 -e th a n e su lp h o n ic a c id , pH a d ju sted to 7 .0 5 w ith NaOH).

The amount o f fragm ent p resen t per ml was th a t gen era ted

from 1 Ag i n t a c t w ild type DNA. 2 M CaClg was added to a

f i n a l c o n c e n tr a t io n o f 0 .1 3 M, and 0 .5 ml sam ples added to

a lm ost c o n f lu e n t dra in ed BHK c e l l m on o layers. The d is h e s

were in cu b a ted a t 3 8 .5 ° from the tim e o f in f e c t i o n , 4 ml

EG5 b e in g added a f t e r 45 m in. At 4 hr p . i . th e c e l l s were

washed w ith E a g le 1s medium and exposed to 1 ml 25# v o l /v o l DMSO* ■■■» ^

i n HeBS fo r 5 min a t room tem p eratu re. The c e l l s were

c a r e f u l ly washed tw ic e w ith E a g le ’ s medium and in cu b a ted a t_ r

3 8 .5 in 4 ml EHu5 f o r 3 d a y s . Then th e m onolayers were

washed tw ic e w ith 4 ml PBSCa, 1 ml PBSCa added, and p la q u es

p ic k e d .

P rep a ra tio n o f HSV DNA

C on flu en t m onolayers o f BHK c e l l s i n 80 oz r o l l e r

b o t t l e s were in f e c t e d a s d e sc r ib ed and in cu b a ted a t 31°•

The in fe c te d c e l l s were detached by a g i t a t io n a f t e r 2 -3 days

and p e l le t e d by c e n tr ifu g a t io n a t 2000 r .p .m . fo r 10 m in.

The medium was r e ta in e d in th e c a se o f HSY-1. The c e l l s

were resuspended in RSB (0 .0 1 M T ris-H C l pH 7*4 , 0 .0 1 M

KC1, 0 .0 0 1 5 M MgCl2 ) c o n ta in in g 0 .5 # v o l /v o l N on id et P40• * _ •

(NP40) and k ep t on i c e fo r 10 m in. N u c le i were p e l l e t e d

by c e n tr i fu g a t io n a t 2000 r .p .m . fo r 5 min and th e

cy to p la sm ic su p ern atan t r e ta in e d . The n u c le i were r e

e x tr a c te d w ith 0 .5 # NP40 in RSB and the second c y to p la sm ic

su pern atant r e ta in e d . V irus was p e l le t e d by c e n tr ifu g a t io n

o f the combined su p ern a ta n ts a t 12000 r .p .m . fo r 3 hr i n an

MSE 6 x 250 ml r o to r .

The p e l l e t was resuspended in NTE, so n ic a te d in an

u ltr a s o n ic bath , 20# w t /v o l sodium d od ecy l su lp h a te (SDS)

- 46 -

added to a f i n a l c o n c e n tr a tio n o f 0 * 5 and e x tr a c te d $$ th

two volum es o f N T E -eq u ilib rated r e d i s t i l l e d phenol by g e n t le

a g i t a t io n on a r o p k -a n d -r o lle r . The upper aqueous phase

was removed and r e -e x tr a c te d w ith two fu r th e r volum es o f

N T E -eq u ilib rated phenol a f t e r c e n tr ifu g a t io n a t 2000 r .p .m .

fo r 10 m in. A fte r a second c e n tr ifu g a t io n s te p the upper

phase was e x tr a c te d f o r 10 min w ith isoam yl a lc o h o l in

chloroform (1 :2 4 v o l /v o l ) and a g a in c e n tr ifu g e d . The upper

aqueous phase c o n ta in in g HSV DNA was d ia ly s e d o v e rn ig h t a t

4° a g a in s t 0 .1 x SSC, and then sto r ed a t - 2 0 ° .

In some in s ta n c e s HSV DNA was p u r if ie d fu r th e r by3

iso p y k n ic banding on caesium c h lo r id e g r a d ie n ts . S o lid

CsCl was added to th e DNA s o lu t io n to g iv e a r e f r a c t iv e in d ex

o f 1 .4 0 1 -1 .4 0 2 . C e n tr ifu g a tio n was f o r 3 days a t 36000 r .p .m .

in a Beckman Ti50 r o to r a t 1 5 ° , or f o r 24 hr a t 40000 r .p .m .

in a S o r v a ll T7865B v e r t i c a l r o to r a t 15° • The g r a d ie n ts* •

were fr a c t io n a te d by p ie r c in g th e tube and c o l l e c t i n g d rop s.

5 / a sam ples o f each f r a c t io n were assayed by e le c tr o p h o r e s is

on a 1 $ ag a ro se g e l c o n ta in in g 0 . 5 yug/ml eth id ium bromi d e >

and n u c le ic a c id s v i s u a l i s e d under lo n g w avelen gth UV

r a d ia t io n (365 nm). F r a c tio n s c o n ta in in g HSV DNA, which

i s c le a r ly d is t in g u is h e d from degraded c e l lu l a r DNA and

RNA, were com bined, d ia ly s e d o v ern ig h t a g a in s t 0 .1 x SSC

a t 4 ° , and s to r e d a t - 2 0 ° .

HSV DNA s u i ta b le fo r n ic k t r a n s la t io n was prepared by

the method o f W ilk ie and C o r t in i (1976^. l in e a r CsCl

g r a d ie n ts o f r e f r a c t iv e in d ic e s 1 .3 8 (bottom ) to 1 .3 6 ( t o p ) ,

c o n ta in in g 20 yt*g/ml eth id ium bromide, were prepared .

0 .2 - 0 .3 ml o f p h e n o l-e x tr a c te d or iso p y k n ic banded HSV-1

DNA a t 100-500 ^Ag/ml was la y e r e d onto each g r a d ien t and th e

- 47 -

tu b es c e n tr ifu g e d a t 40000 r .p .m . fo r 110 min in a Beckman

SW50.1 r o to r . The band o f p u r if ie d v ir u s DNA was v i s u a l i s e d

u s in g a lo n g w avelen gth (365 nm) UV M in er a iig h t handlamp,

and removed v ia an 18-gauge sy r in g e n e e d le . The DNA

s o lu t io n was sa tu r a te d w ith CsCl and e x tr a c te d tw ic e w ith

aqueous p r o p a n -2 -o l sa tu ra te d w ith CsCl to remove eth id ium

brom ide. The low er aqueous phase was d ia ly s e d a g a in s t NTE

and p r e c ip i ta t e d w ith two volum es o f e th a n o l a t - 2 0 ° . The

DNA p e l l e t was r e d is s o lv e d in a sm all volume o f 0 .1 x SSC

and sto r e d a t -2 0 .

P rep a ra tio n o f PRV DNA

BHK c e l l m onolayers in 80 oz r o l l e r b o t t l e s were

in f e c t e d w ith PRV a t a m .o . i . o f 1 in 300 and in cu b a ted a t

37° u n t i l com p lete c . p . e . was ob served . V iru s was h a rv ested

by th e method o f B en-Porat e t a l . (1 9 7 4 ) . The in f e c t e d c e l l

medium was c e n tr ifu g e d a t 5000 r .p .m . fo r 10 min in a

S o r v a ll GSA r o to r , and v ir u s in th e su p ern atan t was

sedim ented onto a 30# w t /v o l su cro se ‘’cu sh io n 11 by c e n tr ifu g a t io n

a t 13500 r .p .m . fo r 1 hr in a S o r v a ll AH627 r o to r . The

v ir u s was la y e r e d onto a l in e a r su cro se g r a d ie n t

(1 5 -3 0 # w t /v o l su cro se in TBSA: 0 .0 1 M T ris-H C l pH 7 .5i - . . .

c o n ta in in g 8 g /1 NaCl, 0 .2 g /1 KC1, 0 .1 g /1 MgC^^HgO,

0 .1 g /1 C a C ^ .i^ O and 10 g /1 bovine serum album in) and

c e n tr ifu g e d in a S o r v a ll AH627 r o to r a t 10000 r .p .m . fo r

140 m in. The v i s i b l e v ir u s band was removed by p ie r c in g th e

tube w ith an 18-gauge sy r in g e n e e d le , d i lu te d s ix f o ld w ith

TBSA and c e n tr ifu g e d a t 13500 r .p .m . fo r 1 hr in a S o r v a ll

AII650 r o to r . The v ir u s p e l l e t was resuspended in 5-10 ml

NTE by s o n ic a t io n , and DNA prepared by phenol e x tr a c t io n

a3 d e sc r ib ed f o r HSV.

P rep a ra tio n o f b acter iop h age lambda DNA

E sc h e r ic h ia c o l i m e t 'r 'n f ( cI q^ S 7) c o n ta in s a lambda

prophage which i s h ea t in d u c ib le and l y s i s d e f i c i e n t . A

s t a r t e r c u ltu r e in TP broth was grown a t 31° and in o c u la te d

in to a u to c la v ed H -broth (5 g /1 D ifco b actop ep ton e, 1 g /1

g lu c o s e , 5 g /1 NaCI, 8 g ? l D ifco n u tr ie n t b ro th , pH 7«2)

a t a d i lu t io n o f 1 :5 0 . The b a c te r ia were grown a t 31° in

a Gallenkamp o r b i t a l in c u b a to r . At a c e l l d e n s ity o fQ

2 -4 x 10 per ml th e c u ltu r e tem perature was in c r e a se d to

42° by th e a d d it io n o f b o i l in g H -broth, and fu r th e r

in c u b a tio n was a t 42° fo r 10 m in. The c u ltu r e temperature*

was then reduced to 37° fo r 1 -2 h r . The b a c te r ia were

c e n tr ifu g e d a t 8000 r .p .m . fo r 2 min in an MSE 6 x 250 ml

r o to r and resuspended in 20 -50 ml T2 b u ffe r (0 .0 5 M Na2HP04 ,

0 .022 M KH2P04 , 0 .0 8 5 M NaCI, 0 .0 0 1 M MgS04 ,~ 0 .0 0 0 1 M CaCl2 ,

0 .0 0 1 # w t /v o l g e l a t i n ) . Chloroform was added to a f i n a l

c o n c e n tr a tio n o f 1 # v o l /v o l to ly s e th e c e l l s , then DNase

to 2 mg/ml. To each 10 ml o f su sp en sio n 0 .7 1 g p o ly e th y le n e

g ly c o l 6000, 0 .1 9 2 g NaCl and 0 .0 2 3 g d ex tran su lp h a te were

added w ith s t i r r i n g , and t h i s was l e f t o v e rn ig h t a t 4° to

a llo w the b acter iop h age to p r e c ip i t a t e . The m a jo r ity o f th e

upper phase was removed*and th e rem ainder c e n tr ifu g e d a t

2000 r .p .m . f o r 5 min. The m a ter ia l a t th e in t e r f a c e was

removed and resuspended in about 2 ml o f 1 # dextran su lp h a te ,

adding 0 .1 5 ml 3 M KC1 per ml o f su sp en s io n . A fte r 2 hr a t

4° th e su sp en sio n was c e n tr ifu g e d a t 2000 r .p .m . fo r 5 min,

and th e su p ern atan t c o n ta in in g th e v ir u s removed. The v ir u s

was p u r if ie d by c e n tr ifu g a t io n on a d is c o n tin u o u s caesium

c h lo r id e g r a d ie n t ( s p e c i f i c d e n s i t i e s o f 1 .7 , 1*5 and 1 .3

g/m l o f CsCl in 0 .0 0 5 M Tris-H Cl pH 7 .2 , 0 .0 0 5 M MgS04 ) a t

35000 r .p .m , fo r 1 hr in a Beckman SW50.1 r o to r . The band

- 49 -

o f v ir u s , in th e phase o f s p e c i f i c d e n s ity 1.5> was d ia ly s e d

a g a in s t 0 .5 x T2 b u ffe r . B acteriop hage DNA was prepared tjy

ph en ol e x tr a c t io n a s d escr ib ed fo r HSV.

E stim a tio n o f DMA c o n c e n tr a t io n s ^

Approximate e s t im a t io n s o f DNA c o n c e n tr a t io n s were

o b ta in ed by v i s u a l com parison o f sam ples w ith known amounts

o f DNA on e th id iu m -sta in e d agarose g e l s under UV i l lu m in a t io n

(365 nm). DNA c o n c e n tr a t io n s o f RNA-free sam ples were more- .rt

a c c u r a te ly e stim a te d by o p t ic a l d e n s ity measurement a t

260 nm, assum ing th a t an o p t ic a l d e n s ity o f 1 .0 corresp ond s

to a DNA c o n c e n tr a t io n o f 40 yug/m l. DNA c o n c e n tr a t io n s o f

sam ples a ls o c o n ta in in g RNA were e stim a te d by th e method

o f H i l l and Whatley (1975)* A s o lu t io n o f m ithram ycin a t

200 yvg/ml was made in 0 .3 M M gC^. 15 yul was mixed w ith

0 .3 ml DNA s o lu t io n and f lu o r e s c e n c e measured a t 550 nm upon

e x c i t a t io n a t 440 nm. DNA o f known c o n c e n tr a t io n , e i t h e r

lambda or c a l f thymus DNA, was used to c o n s tr u c t a standard

curve in th e range 0 -10 /4g/m l.

Pu r i f i c a t io n o f r e s t r i c t i o n en d o n u clea ses

S e v e r a l o f th e r e s t r i c t i o n en d o n u clea ses used in th e

i n i t i a l s ta g e s o f t h i s work were prepared from b a c te r ia l

c e l l p a s te s (T able 3 1 .1 ) . B r a in -h e a rt in f u s io n (BHI) a t

37 g /1 was used f o r bulk p r e p a r a tio n s , which were seeded a t

a d i lu t io n o f 1 :5 0 or 1:100 and grown in a Gallenkamp

o r b i t a l in cu b a to r a t 200-300 r .p .m . B a c te r ia were p e l le t e d

a t e a r ly s ta t io n a r y phase by c e n tr i fu g a t io n in a 6 x 250 ml

MSE r o to r a t 8000 r .p .m . fo r 2 min. They were resuspended

in a sm all volume o f BHI and fr o z e n a t - 2 0 ° .

10-20 g o f c e l l p a s te was hom ogenised in a S e r v a ll

Omnimixer blade hom ogeniser w ith an equal volume o f a c id -

- 50 -

washed g la s s beads (150-200 yum). H om ogenisation was perform ed

on i c e fo r 6 x 2 min p e r io d s a t 10000 r .p .m . w ith 2 min

in t e r v a l s , and th e e x te n t o f hom ogen isation was checked by

m icro sco p ic exam in ation . The g la s s beads were p e l le t e d by

c e n tr i fu g a t io n a t 1000 r .p .m . fo r 5 min, and the su p ern atant

c e n tr ifu g e d a t 40u0Q r .p .ra . fo r 1 hr in a Beckman SW40Ti r o to r

The su p ern atan t was made 1 M in NaCI, 10?° v o l /v o l in g ly c e r o l4 _

and im m ed iate ly su b jec ted to column chrom atography.

The fo l lo w in g column chrom atographic' p roced u res were

em ployed.

a) M olecu lar s i e v e . P r o te in s were sep a ra ted a cco rd in g to

m olecu lar w eight by p a ss in g th e c e l l e x tr a c t through a 2 .5 x

100 era column o f U ltr o g e l AcA34 a t 4 ° . The e lu t in g b u ffe r

(CB) was 0 .02 M T ris-H C l pH 7 .5 , 0 .0 0 1 M EDTA, 0 .0 1 M

2 -m erca p to eth a n o l, 10^ v o l /v o l g ly c e r o l , and an LKB f r a c t io n

c o l l e c t o r was used w ith a flo w r a te o f 2 0 -50 m l/h r . 5 yul

sam ples o f a l t e r n a te 6 ml f r a c t io n s were a ssa y ed fo r n u c le a se

a c t i v i t y as d e sc r ib ed below, and f r a c t io n s c o n ta in in g

r e s t r i c t i o n en d on uclease a c t i v i t y were p o o led and d ia ly s e d

a g a in s t CB.

b) Ion exchange. D iam inoethyl c e l l u l o s e (DE52) or B iorexA 1

70 colum ns o f 5 -20 ml bed volume were poured i n 20 ml sy r in g e

b a r r e ls and e q u il ib r a te d w ith CB. D ia ly se d U ltr o g e l f r a c t io n s

were a p p lie d , washed w ith CB, and e lu te d w ith KC1 g r a d ie n ts•»

in CB. G rad ients o f 0 - 0 .6 M KC1 were r o u t in e ly u sed .

A gain , 5 / * a l iq u o t s o f a lt e r n a te 3 ml f r a c t io n s were a ssayed

fo r n u c le a se a c t i v i t y , and f r a c t io n s c o n ta in in g r e s t r i c t i o n

en d on u clease a c t i v i t y were d ia ly se d a g a in s t CB. Both ty p e s

o f io n exchange column were used f o r m ost en d o n u clea ses

(T able H I.2 ) . The enzymes were co n cen tra ted by a p p lic a t io n .

- 51 -

to sm all B iorex columns (2 -5 m l) , e lu t io n w ith 10 ml o f

1 M KC1 in CB, and d i a l y s i s a g a in s t 50$ v o l /v o l g ly c e r o l in * *

CB. 1 $ w t /v o l s t e r i l e bovine serum album in was added to *

th e enzyme to a f i n a l c o n c e n tr a tio n o f 50 ywg/ml, and s to r a g e

was a t - 2 0 ° .

R e s t r i c t i o n e n d o n u c le a se d i g e s t i o n

A ll r e s t r i c t i o n d ig e s t s were c a r r ie d out in 0 .0 0 6 M

T ris-H C l pH '7.5, 0 .0 0 6 M MgC^, 0 .0 0 6 M 2 -m ercap toeth an o l,

0 .0 2 M KC1, and 0 .0 2 or 0 .1 $ w t /v o l s t e r i l e bovine serum , *• —

album in. R e s t r ic t io n s i t e s are g iv e n in T able EL.4 .

In the p rep a ra tio n o f r e s t r i c t i o n e n d o n u c lea ses , 5 jjOl

a l iq u o t s o f a lt e r n a te f r a c t io n s were in cu b ated a t 37° fo r

3 hr w ith 0 .5 yug lambda DNA in 50 yul r e s t r i c t i o n b u ffe r .

P u r if ie d en d on u cleases were t i t r a t e d a g a in s t 1 yug lambda

DNA in 5 0 1 fo r 3 hr a t 3 7 ° , in order to determ ine th e

amount o f enzyme req u ired fo r com plete d ig e s t io n o f th e DNA.

DNA, e it h e r u n la b e lle d or n ic k t r a n s la t e d , was in cu b ated

a t 10-100 yug/ml fo r 1 -3 hr a t 37° w ith th e req u ired amount

o f enzyme. TagI. BstNI and B e ll d ig e s t io n s were done a t

65°• 0 .1 5 yug lambda DNA was added to r e s t r i c t i o n d ig e s t s

c o n ta in in g sm all amounts o f in v iv o v P - la b e l le d HSV DNA

or r e s t r i c t i o n fragm ents i s o la t e d from n ic k tr a n s la te d HSV

DNA.

R e s tr ic t io n d ig e s t s were term in ated by th e a d d it io n

o f 10 yul o f d y e - P ic o l l , which co n ta in ed 10$ w t /v o l F ic o l l

400, 0 .1 M EDTA and 0 .1 $ w t /v o l bromophenol b lue in 5 x E

b u f f e r ( s e e b e lo w ) .

Agarose g e l e l e c t r o p h o r e s i s

Agarose g e l c o n c e n tr a t io n s between 0 .4 and 2 .0 $ w t /v o l

were em ployed, in E b u ffer (0 .0 3 6 M Trizma, 0 ,0 3 6 M Nai^PO^,

- 52 -

0 .001 M EDTA, pH 7 .8 ) c o n ta in in g 0 .5 yUg/ml eth id ium brom ide.

The agarose was d is s o lv e d by b o i l in g in a Tappan microwave

oven and c o o le d to about 50° b efore p o u rin g . H o rizo n ta l

g e l s were r o u t in e ly u sed , and DHA sam ples w ith added d y e -

P ic o l l were su b je c te d to e le c tr o p h o r e s is a t 2 V/cm fo r

1 6 -2 0 hr a t room tem perature in E b u ffe r c o n ta in in g 0 .5 1

eth id ium brom ide. G els were photographed under lo n g w avelen gth

(365 nm) UV ir r a d ia t io n u s in g P o la r o id ty p e 105 or ty p e 107C

f i lm . G els fo r autorad iograph y were d r ie d on p la in g la s s

p la t e s in an oven a t 6 0 ° .

P o lyacry lam id e g e l e le c tr o p h o r e s is

A s to c k s o lu t io n o f 29 > w t /v o l acry lam id e and 1 # w t /v o lm — <

N N '-m eth y len eb isacry lam id e in w ater was d i lu te d to th e

req u ired c o n c e n tr a tio n w ith th e a d d it io n o f 10 x THE to a

f i n a l c o n c e n tr a tio n o f 1 x TBE (0 .0 8 9 M Trizma, 0 .0 8 9 M b o r ic

a c id , 0 .0 0 2 5 M EDTA) and 10?* w t/v o l ammonium p ersu lp h a te toT ■ . *

0 . 0 6 7 6 0 ml o f t h i s s o lu t io n was d egassed u s in g a vacuum

pump and 30 yul N ,N ,N *,N f -te tr a m e th y le th y le n e d ia m in e (TEMED)

added, and th e g e l poured (265 mm x 165 mm x 1 .5 mm).

E le c tr o p h o r e s is was c a r r ie d ou t in 1 x TBE a t 3 V/cm fo r

10-20 hr a t room tem p erature, and th e g e l s were d r ied on

Whatman 3MM paper under vacuum in a B iorad h eated g e l d r ie r .

G eneral l a b e l l i n g o f DNA w ith ^2P

N ick t r a n s la t io n ’o f DNA i n , v i t r o was done e s s e n t i a l ly

a cco rd in g to th e method o f Rigby e t a l . (1 9 7 7 ) . 180 pm oles

o f each o f * - 52P dATP, dGTP, dCTP and dTTP (350 Ci/mmole), . . ^ —•

were added to a tube and d r ied in an Edwards l y o p h i l i s e r .

1 yug DNA in 100 yul n ic k t r a n s la t io n b u ffe r (0 .0 5 M Tris-H C l

pH 7 .8 , 0 .0 0 5 M MgC^, 58 yug/ml bovine serum album in, 0 .0 0 5 M

- 53 -

2-m ercap toeth an o l) was added to th e tube on i c e . 2*5 u n i t s

DNA polym erase I were added and tube in cu b ated a t 1 4 -1 5 °

f o r 90 m in. DNA p o s s e s s in g few er n ic k s than HSV DNA, suoh

a s th a t from b acter iop h age lambda and $X174, or piasm id DNA,— 3 m m /L '

was in cu b ated w ith th e a d d it io n o f DNase to 10 J or 10 m g/m l.

Iso to p e in c o r p o r a tio n was m onitored by s p o t t in g 2 yul sam ples

onto f i l t e r d i s c s and p r e c ip i t a t in g th e DNA w ith 5# w t /v o li *

t r i c h lo r a c e t i c a c id f o r 15 min on i c a . A fte r w ashing in

e th a n o l then a ce to n e th e d i s c s were d r ied and counted f o r

^2P in s c i n t i l l a n t . T h is method produced n ic k tr a n s la te d' 8DNA o f s p e c i f i c a c t i v i t y 1 -2 x 10 c .p .m ./iu g , maximal

in c o r p o r a tio n (38-70/*) b e in g reached a t 90 m in. The n ic k

t r a n s la t io n r e a c t io n was term in ated e i t h e r by adding EDTA

to a f i n a l c o n c e n tr a tio n o f 0 .0 1 M fo llo w e d by h e a t in g 1a t

65° fo r 10 min, or by phenol e x tr a c t io n . L a b e lled DNA was

sep arated from u n in corp orated d eo x y n u c leo sid e tr ip h o sp h a te s

by e lu t io n through a 15 cm column o f Sephadex G50 in NTE.32HSV DNA was P - la b e l le d in v iv o a s d e sc r ib ed by P resto n

n ' '■ ' ' - ~ 6e t a l . (1 9 7 8 ). 50 mm P e tr i d is h e s were seed ed w ith 2 x 10

c e l l s / p l a t e in E C l-P i and in cu b a ted a t 37* o v e r n ig h t . V irus

was added in 0 .2 ml E C l-P i a t a m .o . i . o f 5 p . f . u . / c e l l ,

in cu b ated a t 31° fo r 1 h r , th e m onolayers washed tw ic e w ith

E C l-P i, and 2 ml E C l-P i added. A fte r a fu r th e r 2 -3 hr a t

3 1 ° , 0 .2 5 - 0 .5 mCi o f c a r r ie r - f r e e ^2P -orth op h osp h ate was

added in 0 .5 ml E C l-P i, and the p la t e s were in cu b a ted a t 31°

f o r 2—3 d a y s . The in f e c t e d c e l l s were removed w ith a rubber

policem an or bent P a steu r p ip e t t e and c e n tr ifu g e d a t 2000

r .p .m . fo r 5 m in. Each c e l l p e l l e t was e x tr a c te d once w ith

5 ml 0*5/^ NP40 in RSB. The cy to p la sm ic su p ern atan t was4

combined w ith th e medium and c e n tr ifu g e d a t 3 5 0 0 0 r .p .m .

fo r 1 hr a t 4° in an Oakridge tube in a Beckman T i50 r o to r .

- 54 -The v ir u s p e l l e t was resuspended by s o n ic a t io n in 0 .5 ml

NTE, th en 50 yul 25?* w t /v o l sodium d od ecy l su lp h a te and

0 .5 ml N T E -saturated phenol were added to th e v ir u s su sp en sion

in a 1 .5 ml Eppendorf capped p l a s t i c tu b e . A fte r g e n t le

a g i t a t io n th e tube was c e n tr ifu g e d fo r 5 min a t 2000 r .p .m .,

and th e upper phase removed and e x tr a c te d w ith 0 .5 ml

isoam yl a lc o h o l in ch loroform ( 1 : 2 4 ) . B o iled RNase A and T1

were added to th e removed upper phase to f i n a l c o n c e n tr a t io n s

o f 25 yug/ml and 50 U/ml r e s p e c t iv e ly , and th e DNA d ia ly s e d

o v e r n ig h t a g a in s t 0 .1 x SSC a t room tem p eratu re. 10 yul

sam ples were s c i n t i l l a t i o n counted i n 2 ml w ater in order

to e q u a lis e co u n ts b efore r e s t r i c t i o n and g e l a n a ly s is .

I s o la t i o n o f r e s t r i c t i o n fragm ents

The method o f Wil'kie and C o r tin i (1976) was used to

i s o l a t e r e s t r i c t i o n fragm ents from agarose g e l s . U n la b e lled

fragm ents were lo c a te d by s ta in in g e i t h e r th e whole

h o r iz o n ta l g e l or a s t r ip from each edge w ith 0 . 5 yug/ml

eth id ium bromide in E b u ffe r , and n ic k tr a n s la te d fragm ents

were e x c is e d from agarose tube g e l s ( 1 .5 x 18 cm) which had

been e le c tr o p h o r e se d in th e p resen ce o f 0 .5 >ug/ml eth idium

brom ide. Agarose fragm ents were d is s o lv e d i n 2 -10 ml 5 M

sodium p e r c h lo r a te a t 60° and a p p lied to h y d ro x y la p a tite

columns o f bed volum es 0 .2 - 1 .0 ml in sy r in g e b a r r e ls .

A p p lic a tio n and e lu t io n o f th e DNA were done a t 6 0 ° . The

colum ns were washed w ith 5 x 1 ml 5 M sodium p e r c h lo r a te and

then w ith 2 x 1 ml 0 .1 4 M sodium phosphate pH 6 .8 . Double

strand ed DNA was e lu te d w ith 4 x 0 .5 ml 0 .4 M sodium

phosphate pH 6 .8 , and d ia ly s e d o v e rn ig h t a g a in s t 0 .1 x SSC.

R a d io a c tiv e sam ples were s c i n t i l l a t i o n counted f o r32Cerenkov r a d ia t io n from P.

- 55 -

T ran sfer o f DNA fragm ents to n i t r o c e l lu lo s e

The b lo t t in g tech n iq u e o f Southern (1975) in v o lv e s

th e im m o b ilisa t io n o f s in g le stranded DNA on a n i t r o c e l lu lo s e

f i l t e r . R e s tr ic te d HSV DNA was e le c t r o p h o r e t ic a l ly

sep ara ted on an agarose g e l (betw een 0 .4 and 1 .0 $ w t /v o l)

in a s in g le s l o t spanning th e g e l . The g e l was soaked f o r

1 hr in g e l soak I (0*2 M NaOH, 1 M NaCI) and th en f o r 1 hr

in g e l soak I I (1 M T ris-H C l pH 8 , 1 M N aC I). The g e l was

tr a n s fe r r e d to two s h e e ts o f Whatman 3MM paper on a g la s s

p la t e supported in a tr a y o f 2 1 o f 6 x SSC. A sh e e t o f

m oistened n i t r o c e l l u l o s e th en a sh e e t o f 3MM paper were

a p p lie d to th e g e l su r fa c e , fo llo w e d by a w eighted s ta c k o f

c u t paper to w e ls . T ran sfer o f th e s in g le strand ed DNA to

th e n i t r o c e l lu lo s e was a llow ed to occu r o v e rn ig h t a t room

tem p eratu re. The n i t r o c e l lu lo s e sh e e t was washed in 3 x SSC,

d r ied in a i r , then baked in a vacuum oven a t 70° fo r 2 h r .

I t was th en used e i t h e r fo r s t r ip o r c r o s s - b lo t h y b r id is a t io n .

B lo t h y b r id is a t io n

H y b r id isa tio n a t 75° in aqueous s o lu t io n (W ilk ie , 1976)

or a t 45° in 50>* v o l /v o l formamide (McConaughy e t a l . , 1969)

was u sed . N ick tr a n s la te d probe DNA was denatured by h e a tin g

a t 100° fo r 5 min in 0 .1 M NaOH, and th en n e u tr a l is e d on i c e

w ith 1 M HC1 a f t e r th e a d d it io n o f 1 M T ris-H C l pH 7*5 to

a c o n c e n tr a tio n o f 0 .0 5 M. 10^-10^ c .p .m . o f ^2P-DNA was

r o u t in e ly used per h y b r id is a t io n . 5 mm s t r i p s were cu t from

a n i t r o c e l lu lo s e b lo t and in cu b ated in a sh ak in g w ater bath

a t 75° fo r 1 hr in 15 ml p l a s t i c screw -top tu b es c o n ta in in g

5 ml o f 3 x SSC and D enhardt1s s o lu t io n (D enhardt, 1966;

0.02>* w t /v o l F ic o l l 400, 0 .0 2 $ w t/v o l p o ly v in y lp y r r o lid o n e ,* 4

0 .0 2 $ w t /v o l bovine serum alb u m in ). H y b r id isa t io n s c a r r ie d

out in 5 0 $ formamide in 3 x SSC and Denhardt*s s o lu t io n were

- 56 -

in cu b ated a t 45°• The tu b es were drained and th e denatured

probe added a f t e r b ein g made up to 5 ml i n 3 x SSC and

D enhardt1s s o lu t io n , or 50$ formamide in 3 x SSC and D enhardtf s

s o lu t io n . H y b r id isa tio n was a llow ed to occu r f o r 2 -4 days

a t 75° or 45° i n a shaking w ater bath , and th en th e s t r i p s

were washed w ith 2 x SSC a t 60° f o r a t l e a s t s i x hours w ith

se v e r a l changes o f s o lu t io n . The s t r i p s were d r ied in a i r ,

a p p lie d to a g la s s p la t e w ith d o u b le -s id e d S e l lo ta p e , baked

a t 70° fo r 30 min, and autorad iograp h ed .

The method o f c r o s s - b lo t h y b r id is a t io n , d evelop ed by

H utchison (S a to e t g A ., 1 9 7 7 ), in v o lv e s th e sim u ltan eou s

t r a n s fe r o f denatured n ic k tr a n s la te d DNA fragm ents from an

agarose g e l to a n i t r o c e l lu lo s e sh e e t c o n ta in in g im m ob ilised

u n la b e lle d DNA fra g m en ts . H y b r id isa tio n o ccu rs in s i t u ,i ■» ■ "

and homology between fragm ents i s d e te c te d a s s p o ts on an

a u torad iograp h . A square, n i t r o c e l lu lo s e b lo t was prepared

a s d e sc r ib ed and soaked fo r 1 hr a t 75° in 4 x SSC and

0 .1 $ w t /v o l sodium d od ecy l su lp h a te . 1 -2 /*g n ic k tr a n s la te d

HSV DNA was r e s t r i c t e d and e le c t r o p h o r e t ic a l ly sep a ra ted on

an agarose g e l o f e q u iv a le n t d im ensions to th e f i r s t g e l , and

then au torad iograp h ed . The g e l was tr e a te d w ith g e l soak I

then I I , and DNA fragm ents tr a n s fe r r e d to th e n i t r o c e l lu lo s e

sh e e t turned through 90° so th a t the DNA bands on th e sh e e t and

on th e g e l were p e r p e n d ic u la r . T ran sfer was c a r r ie d ou t

o v ern ig h t in 4 x SSC and 0 .1 $ sodium d od ecy l su lp h a te a t 75°

in an oven . A ll components o f th e system e x cep t th e g e l were

heated to 75° before commencing t r a n s f e r . The n i t r o c e l lu lo s e

sh e e t was th en washed e x te n s iv e ly w ith 3 x SSC a t 6 0 ° . In

order to o r ie n ta t e th e u n la b e lle d d im ension , a s t r ip was

removed from th e sh e e t b efore c r o s s - b lo t t in g and h y b r id ised

- 57 -

w ith 10^ c .p .m . o f u n r e s tr ic te d n ic k tr a n s la te d HSV DNA.

The s t r ip was washed w ith the sh e e t and reassem b led a lo n g s id e

i t . The assem bly was autoradiographed a f t e r d ry in g and

b a lin g . The la b e l le d dim ension was o r ie n ta te d lay comparing

th e autorad iograp h o f th e c r o s s - b lo t w ith th a t o f th e wet g e l

c o n ta in in g th e n ic k tr a n s la te d fragm en ts .

Autoradiography

G els were autoradiographed e i t h e r a t room tem perature

w ith Kodak K odirex f i lm or w ith Kodak X-Omat H f i lm , or a t

-7 0 ° w ith p r e fla sh e d Kodak X-Oraat f i lm and a Dupont Cronex

p h osp h otu n gsta te sc r e e n ,

R e s t r ic t io n fragm ent nom enclature

Gel e le c tr o p h o r e s is o f r e s t r i c t e d DNA g iv e s a*?

c h a r a c t e r i s t i c number o f bands which are sep ara ted a cco rd in g

to m o lecu lar w e ig h t. Bands are g iv e n l e t t e r s a lp h a b e t ic a l ly

or numbers s e q u e n t ia l ly from slow er to f a s t e r m ig ra tin g

bands ( la r g e r to sm a ller fr a g m e n ts ) . T h is a ls o a p p lie s to „

bands which are l e s s than m olar, such a s th o se gen erated

from th e ter m in a l and j o in t fragm ents o f HSV DNA. The o n ly

exceptions to t h i s are bands r e s u l t in g from sequence a d d it io n s

to o th e r fra g m en ts . For exam ple, th e jo in t fragm en ts HSV-1

BamHI k and IISV-2 BamHI g c o n s i s t o f HSV-1 BamHI k and HSV-2

BamHI g , r e s p e c t iv e ly , w ith an a d d it io n a l a sequence ( s e e ,

fo r exam ple, F igu re C 1 .3 ) . A band which i s more than m olar,

and y e t r e p r e s e n ts o n ly one sequence because th e c o n s t i tu e n t

fragm ents o r ig in a te from e n t ir e ly w ith in a r e p e a t seq u en ce,

i s g iv en o n ly one d e s ig n a tio n ( e . g . HSV-2 BamHI p , g ' , mf

in F igu re C l .2 0 ) . Fragments which com igrate in th e g e l

system used are d esig n a ted from l e f t to r ig h t on th e genome

map ( e .g . HSV-2 BamHI h ij in F igu re C 1 .2 o ).

- 58 -

C a lc u la t io n o f r e l a t iv e m o la r it ie s o f r e s t r i c t i o n fragm ents

A utoradiographs were scanned u s in g a J o y c e - lo e b l

a u to d e n s ito m e te r , and peaks were cu t o u t and w eighed .

M olar r a t i o s o f in d iv id u a l fragm ents were c a lc u la te d from

th e fo l lo w in g form u la .

M olar r a t io — wt o f peak__________ ^ m .w t. o f genome__t o t a l wt o f peak8 m .wt. -of^ fragm ent

R a tio n a le f o r mapping r e s t r i c t i o n s i t e s

The purpose o f r e s t r i c t i o n mapping i s to r e la t e the

r e s t r i c t i o n p r o f i l e to th e p h y s ic a l lo c a t io n o f r e s t r i c t i o n

fragm en ts w ith in th e genome. In t h i s stu d y th e fo l lo w in g

m ethods were u sed .

1 . R ecleavage exp erim en ts. In d iv id u a l ^ P - l a b e l l e d

r e s t r i c t i o n fragm en ts were i s o la t e d and r e c le a v e d w ith a

second enzyme, and th e p rod u cts su b jeo ted to e le c tr o p h o r e s is

a lo n g s id e th e d ig e s t o f th e whole genome w ith th e second

enzyme. T h is method shows which fragm ents produced by th e

second enzyme are w ith in th e r e c le a v e d fragm ent (p rod u cts

co rresp o n d in g to bands in th e r e s t r i c t i o n p r o f i l e o f th e

genome w ith th e second enzym e), and which o f th e produ ct

fragm en ts are a t th e ends o f the r e c le a v e d fragm ent (bands

n o t c o r r e sp o n d in g ) . Large p a r ts o f th e p h y s ic a l map fo r

th e second enzyme may then be d e r iv e d , e s p e c ia l ly whsJSfr th e

map f o r th e f i r s t enzyme i s a lrea d y known. T his method may

be r ev e rse d tjy r e c le a v in g fragm ents produced by th e second

en d on u clease w ith the f i r s t .

- 59 -

2 . Double d ig e s t exp erim en ts, P - la b e l le d DNA was c le a v e d

s im u lta n eo u sly w ith two e n d o n u c lea ses . I f a band i s p resen t

in th e s in g le d ig e s t p r o f i l e but n o t in th e double d ig e s t

p r o f i l e , t h i s in d ic a t e s th a t the fragm ent c o n ta in s a r e s t r i c t i o n

s i t e fo r th e second enzyme. The apparent p resen ce o f a band

in th e double d ig e s t does n o t n e c e s s a r i ly show th a t th e fragm ent

was u n c lea v ed , a s a fragm ent o f la r g e r m o lecu la r w eigh t may have

been c le a v ed to g iv e a product which f o r t u i t o u s ly com igrated

w ith th e band under c o n s id e r a t io n .

3 . H y b r id isa tio n exp er im en ts. ^ P - l a b e l l e d i s o la t e d fragm ents

were h y b r id ised to a n i t r o c e l lu lo s e b lo t o f sep a ra ted

r e s t r i c t i o n fragm ents produced by an o th er en d o n u c lea se .

A lte r n a t iv e ly th e c r o s s - b lo t method was u sed , but th e d a ta

o b ta in ed were e s s e n t i a l l y th e same. These m ethods, taken in

c o n ju n ctio n w ith r e c le a v a g e exp erim en ts, show which fragm ents

are w ith in th e complem entary fragm ent and which extend

p a s t i t s en d s.

C ontam ination o f r e s t r i c t i o n fragm ents i s o la t e d from g e ls

D ig e sted n ic k tr a n s la te d DNA produced a background o f

con tam in atin g seq u en ces in p r e p a r a tiv e g e l s , e s p e c ia l ly i n

the h igh m olecu lar w eigh t r e g io n . T h is i s a fe a tu r e o f

th e n ic k t r a n s la t io n r e a c t io n , but was som etim es due to

con tam in ation o f the r e s t r i c t i o n en d on u clease w ith non

s p e c i f i c n u c le a s e s . Bands in c lo s e p ro x im ity to o th e r s

tended to be contam inated w ith th o se s p e c i f i c seq u en ces .

T h erefo re , i s o la t e d fragm ents were e le c t r o p h o r e t ic a l ly

sep arated both u n c lea v ed , and c le a v ed w ith an endon u clease

o f known p h y s ic a l map, in order to ensure id e n t i t y and

e stim a te p u r ity .

- 60 -

P a in t bands on au torad iograp h s were presumed to r e s u l t

e ith e r from g e n e r a l sequence con tam in ation or from proxim al

band con tam in ation ( e . g . in F igu re 0 1 .8 , Kpnl k was n o t

c le a v ed by Hindl l l , but i t was contam inated both g e n e r a l ly ,

and s p e c i f i c a l l y by Kpnl 1 ) . In n e a r ly a l l r e c le a v a g e

experim en ts some o f th e parent fragm ent was r e fr a c to r y to

d ig e s t io n , d e s p ite com plete d ig e s t io n o f c a r r ie r lambda DNA,

and th e ab sen ce o f bands r e s u l t in g from p a r t ia l d ig e s t io n o f

th e fragm en t. The reason f o r t h i s o b se r v a tio n i s unknown.

Sequence con tam in ation was a ls o d e te c te d in h y b r id is a t io n

experim en ts in v o lv in g i s o la t e d n ic k tr a n s la te d fragm en ts.

G eneration o f b a c t e r ia l c lo n e s b earin g recom binant p lasm id s w ith HSV DNA i n s e r t s - - - -

HSV DNA fragm ents were l ig a t e d in to th e v e c to r pAT153

(Twigg and S h e r r a tt , 1 9 8 0 ), which i s a d e le t io n mutant o f

pBR322 unable to undergo co n ju g a l t r a n s fe r and which c a r r ie s

gen es c o n fe r r in g r e s i s ta n c e to t e t r a c y c l in e and a m p ic i l l in .

Recombinant p lasm id s were tr a n s fe c te d in to th e h o st

E sc h e r ic h ia c o l i K12 s t r a in HHL01 (Boyer and R ou llan d -D u sso ix ,

1 9 6 9 ) . The h o s t was grown in L -broth (0 ,1 7 M NaCI, 10 g /1

D ifco b a c to try p to n e , 5 g /1 y e a s t e x tr a c t ) a t 37° in a

Gallenkamp o r b i t a l in c u b a to r . Transformed b a c te r ia b earin g

pAT153 were grown in 1 -b r o th c o n ta in in g 100 ^ug/ml a m p ic i l l in .

S tock s were prepared by p e l l e t in g b a c te r ia from a 20 ml

o v ern ig h t c u ltu r e l?y c e n tr ifu g a t io n a t 8000 r .p .m . f o r 2 min,

and re su sp en d ing in 10 ml 40# v o l /v o l g ly c e r o l in 1 # w t /v o l

D ifco bactopeptone b e fo re s to r in g a t - 2 0 ° . in 1 ml a l iq u o t s .

HSV-2 HindI I I fragm ents were l ig a t e d in t o pAT153

e s s e n t i a l ly by the method o f Tanaka and Weisblum (1975)*

3 /'g HSV-2 s t r a in HG52 DNA and .l /< g pAT 153 DNA were

- 61 -

s e p a r a te ly d ig e s t e d w ith Hind I I I and th en h e a te d a t 7 0 ° f o r

10 min to i n a c t i v a t e th e en d o n u clea se# The two r e a c t io n s

were mixed and a p p r o p r ia te s o lu t io n s added to b r in g th e f i n a l

c o n d it io n s to 0 .0 2 M T ris-H C l pH 7 .5 , 0 .0 1 M MgCl2 , 0 .0 0 0 5 M

ATP, 0 .0 1 M d i t h i o t h r e i t o l , and 10 U/m l T4 DNA l i g a s e i n

a f i n a l volum e o f 50 z*1 , The m ix tu re was in c u b a te d a t 4°

f o r 16 h r .

The j o in t fra g m en ts HSV-1 BamHI k ( s t r a i n s 17 and USA-8)

and HSV-2 BamHI g ( s t r a i n HG52), and a l s o HSV-1 BamHI y

( s t r a i n 1 7 ) , were i s o l a t e d by h y d r o x y la p a t it e chrom atography

(HSV-1 BamHI y was i s o l a t e d by M -J. M urchie) a f t e r

e le c t r o p h o r e s i s o f 100 yug d ig e s t e d DNA on a g a r o se g e l s

i n th e a b sen ce o f e th id iu m brom ide. The fra g m en ts were

l i g a t e d in 20 yul r e a c t io n volum es a s d e s c r ib e d a b o v e , u s in g

betw een 0 .1 and 0 ,5 yug Bam H I-cleaved pATl53 DNA and th e

amount o f HSV fragm en t p o s s e s s in g th r e e to f i v e t im e s a s many

BamHI te r m in i a s th e pAT153 DNA.

L ig a te d m ix tu r e s w ere t r a n s f e c t e d i n t o th e h o s t

b acteriu m e s s e n t i a l l y a s d e s c r ib e d by Cohen e t a l . ( 1 9 7 2 ) ,

a f t e r c h e c k in g l i g a t i o n by g e l e l e c t r o p h o r e s i s a lo n g s id e

u n lig a te d and r e c le a v e d r e a c t io n m ix tu r e s . A l l p r o ce d u r es

in v o lv in g th e u se o f l i v i n g b a c t e r ia su b seq u en t to l i g a t i o n

o f DNA were perform ed under C ategory I I c o n d i t io n s , h a v in g

o b ta in ed th e a p p ro v a l o f th e G en etic M a n ip u la tio n A d v isory

Group. Jg. c o l i KL2 s t r a in HB101 c e l l s w ere grown to an

o p t i c a l d e n s i t y o f 0 .6 a t 630 nm, h a r v e s te d by c e n t r i f u g a t io n

a t 8000 r .p .m . f o r 2 m in, washed by r e su sp e n d in g in 400 ml

0 .0 1 M NaCI and a g a in c e n t r i f u g in g . The p e l l e t was

resu sp en d ed in 400 ml 0 .0 3 M CaCl2 , iiep t on i c e f o r 20 m in,

and h a r v e s te d a s b e fo r e . The b a c te r ia were re su sp en d ed in 9 .4 ml

- 62 -

o f 1 5 $ v o l / v o l g ly c e r o l in U .03 M CaCl2 , and 0 .2 ml a l iq u o t s4 ~ o

s to r e d a t -7 0 . Under C ategory I I co n ta in m en t c o n d it io n s

a l i q u o t s w ere m ixed in 15 ml capped p l a s t i c tu b e s w ith

l i g a t e d DNA sa m p les , k ep t on i c e f o r 30 m in , th en in c u b a te d

a t 37° f o r 5 m in. 3 ml L -b ro th was added and th e tu b e s

a e r a te d by b u b b lin g a t 37° f o r 90 m in . 0 .1 ml a l iq u o t s were

sp read on 90 mm L -b r o th a g a r p l a t e s c o n ta in in g 1 0 0 yug/m l

a m p ic i l l i n , and in c u b a t io n was a t 37° o v e r n ig h t . A p p ro p ria te

c o n t r o l s were in c lu d e d in th e t r a n s f e c t i o n s : u n d ig e s te d pAT153

-DNA, c le a v e d pATl53 DNA, and c le a v e d pATl53 DNA w hich had■* ■» _ • ibeen l i g a t e d .

The h y b r id is a t io n te c h n iq u e o f G ru n ste in and H ogness

(1 9 7 5 ) was u sed to i d e n t i f y c o lo n ie s b ea r in g recom b in an t

p la s m id s . S t e r i l e g r id d ed n i t r o c e l l u l o s e f i l t e r s were b o ile d

t h r ic e i n w a te r , a u to c la v e d and baked . B a c t e r ia l c o lo n ie s

were t r a n s fe r r e d u s in g s t e r i l e c o c k t a i l s t i c k s on to th e g r id s

on L -b ro th a g a r p l a t e s c o n ta in in g 100 yug/ml a m p ic i l l i n , and

a l s o to a m ic r o t i t r e p la t e c o n ta in in g 0 .2 m l/w e l l L -b ro th

p lu s a m p ic i l l i n . G rids were in c u b a te d a t 3 7 ° o v e r n ig h t and

th e m ic r o t i t r e p l a t e s were k ep t a t room te m p e r a tu r e . The

n i t r o c e l l u l o s e f i l t e r s were t r a n s f e r r e d w ith c o lo n ie s

upperm ost i n tu rn a t room tem p era tu re to f i l t e r p a p ers soaked

w ith 0 .1 U HC1 (1 m in ), 0 .5 M NaOH (15 m in ) , 1 M T ris-H C l

pH 7 .5 (5 m in ), and 1 .2 M NaCI (15 m in ), r e s p e c t i v e l y . The

f i l t e r s were d r ie d i n a i r and baked a t 7 0 ° i n vacuo f o r 2 h r .

P r e in c u b a t io n and h y b r id is a t io n o f th e f i l t e r s were c a r r ie d

ou t in 50/* form am ide a t 45° a s d e s c r ib e d f o r b lo t h y b r id is a t io n .

N ick t r a n s la t e d 1ISV-2 DNA was used a s probe f o r HSV-2 Hind i I I

c lo n e s , and th e i n d iv id u a l ly i s o l a t e d r e s t r i c t i o n fr a g m en ts

l o r HSV-1 HamHI k and y and HSV-2 BamHI g . C o lo n ie s b ea r in g

p la sm id s c o n ta in in g IISV DNA i n s e r t s were i d e n t i f i e d by

- 63 -

a u to r a d io g r a p h y . S to c k s were p rep ared from th e m ic r o t i t r e

p la t e c u l t u r e s a f t e r a f u r th e r s te p o f c o lo n y - p u r i f i c a t io n .

The i d e n t i t y o f p la sm id DNA was a s c e r ta in e d t»y h y b r id is a t io n

o f n ic k t r a n s la t e d p la sm id DNA to b lo t s t r i p s o f HSV

r e s t r i c t i o n d i g e s t s , and by r e s t r i c t i o n m apping o f th e p lasm id

in com p arison w ith HSV genome r e s t r i c t i o n m aps.

Between 1 and 1 0 # o f a m p i c i l l i n - r e s i s t a n t c o lo n ie s were

p o s i t i v e f o r HSV DNA i n s e r t s . 37 HSV-2 H in d l l l c lo n e s were

c h a r a c t e r is e d , 8 HSV-1 s t r a in 17 BamHI k c lo n e s , 3 HSV-1

s t r a in USA-8 BamHI k c lo n e s , 9 HSV-2 BamHI g c lo n e s , and 1

HSV-1 s t r a i n 17 BamHI y c lo n e . T able B I .3 shows the e q u iv a le n t

sta n d a rd n o m en cla tu re f o r t r i v i a l names o f th e c lo n e s u sed

i n t h i s work.

P lasm id DNA was i s o l a t e d e s s e n t i a l l y a s d e s c r ib e d by

C le w e ll and H e lin s k i (1 9 7 0 ) , a f t e r l y s i s o f b a c t e r ia w ith

a n e u tr a l d e te r g e n t (Komano and S in sh e im e r , 1 9 6 8 ) . 1 -2 1

o f l - b r o t h c o n t a in in g 100 yi^;/ml a m p ic i l l in was in o c u la te d w ith

an o v e r n ig h t c u ltu r e o f 1 0 -2 0 ml b a c t e r ia , and grown o v e r n ig h t

a t 37° i n a Gallenkamp o r b i t a l in c u b a to r . C hloroform was

added to 1^ v o l / v o l and th e b a c te r ia h a r v e s te d a f t e r 20 min4

by c e n t r i f u g a t io n a t 8000 r .p .m . f o r 2 m in . The p e l l e t was

resu sp en d ed i n 2 5> w t /v o l su c r o se in 0 .0 5 M T ris-H C l pH 8 ,

10 ml o f 5 mg/ml ly sozym e was added, and th e m ix tu re was k ep t

on i c e f o r 30 m in. In c u b a t io n on i c e was co n tin u e d f o r 30

min a f t e r th e a d d i t io n o f 10 ml 0 .2 h EDTA, and th en 133 /*1

NP4-0 was ad d eu . The tu b e was m ixed g e n t ly , k ep t on i c e f o r

10 m in , and c e n tr i fu g e d a t 20000 r .p .m . f o r 90 min in a

S o r v a l l SS34 r o t o r . The su p er n a ta n t was e x tr a c te d tw ic e by

v ig o r o u s sh a k in g w ith N T E -eq u ilib ra ted p h e n o l, e x tr a c te d

once w ith c h lo r o fo rm , and 3 M sodium a c e t a t e pH 7 .5 was added

- 64 -

to a f i n a l c o n c e n tr a t io n o f 0 .3 M. N u c le ic a c id s weref

p r e c ip i t a t e d by th e a d d it io n o f h a l f a volum e o f propan-2-r-ol,

and p e l l e t e d by c e n t r i f u g a t io n a t 2000 r .p .m . f o r 30 min

a f t e r 1 hr a t room te m p e ra tu r e . The p e l l e t was resu sp en d ed

i n 20 ml w a te r , and 2 5 .5 g s o l i d caesiu m c h lo r id e and nil

eth id iu m brom ide a t 10 mg/ml added, and w a ter added to a volum e

o f 34 m l. T h is was d iv id e d betw een two 17 ml tu b e s and

c e n t r i fu g e d f o r 20 hr a t 1 5 ° in a S o r v a l l TV865B v e r t i c a l

r o t o r . The r e s u l t i n g band o f p lasm id DNA i n ea ch tu b e was

v i s u a l i s e d by lo n g wave UV i r r a d i a t i o n (365 nm) and removed

by p ie r c in g th e tu b e w ith a s y r in g e n e e d le . The volume o f

th e DNA s o lu t io n was in c r e a s e d to 17 ml by th e a d d it io n o f

0 .7 5 g /m l caesiu m c h lo r id e c o n ta in in g 0 .5 mg/ml eth id iu m

brom ide. P lasm id DNA was banded a secon d tim e by c e n t r i f u g a t io n

a s d e s c r ib e d a b o v e , removed from th e tu b e v ia a s y r in g e

n e e d le , s a tu r a te d w ith C sC l, and e x tr a c te d tw ic e w ith aqueous

p r o p a n -2 -o l s a tu r a te d w ith CsCl to remove e th id iu m brom ide.

P u r i f ie d p la sm id DNA was d ia ly s e d o v e r n ig h t a g a in s t NTE,n i

e th a n o l p r e c ip i t a t e d , and r e d is s o lv e d in 1 -2 ml 0 .1 x SSC.

One l i t r e o f b a c t e r ia l c u l tu r e g e n e r a l ly y ie ld e d 0 .5 - 1 mg

o f p la sm id DNA.

La b e l l i n g o f DNA te r m in i w ith

R e s t r i c t io n fra g m en ts from c lo n e d DNA w ere la b e l l e d a t

t h e ir 5* te r m in i e s s e n t i a l l y a c c o r d in g to th e method o f

Maxam and G ilb e r t (1 9 8 0 ) . 1 -4 /*g o f a DNA fra g m en t i s o l a t e d

from an a g a r o se g e l was d ig e s t e d in 50 / a1 o f r e s t r i c t i o n

b u ffe r w ith th e r e s t r i c t i o n en d o n u c lea se c le a v in g a t th e

s i t e s to be l a b e l l e d ( s e e T ab le B 1 .4 ) . 50 jXL o f 0 .0 5 M

T ris-H C l pH 8 and 20 U b a c t e r ia l a l x a l i n e p h o sp h a ta seo

were added and in c u b a te d a t 65 f o r 1 h r . The DNA was

e x tr a c te d w ith 100 o f N T E -satu rated p h e n o l, e th a n o l

p r e c i p i t a t e d , washed w ith 9 5 $ v o l / v o l e t h a n o l, and l y o p h i l i s e d .

The DNA was d i s s o lv e d in 20 yul o f 0 .0 5 M T ris-H C l pH 7 .6 ,

0 .0 1 M MgCl2 , 0 .0 0 5 M d i t h i o t h r e i t o l , 0 .0 0 0 1 M sp e r m id in e ,

0 .0 0 0 1 M EDTA. In th e c a s e o f a r e s t r i c t i o n s i t e n o t g iv in g

a p r o tr u d in g 5' te r m in u s , th e r e a c t io n m ix was h e a te d a t 65°

f o r 2 min and quenched on i c e . 5 /*1 y’-^ P -A T P ( 5000 C i/m m ole)

and 20 U T4 p o ly n u c le o t id e k in a se were added and th e

r e a c t io n in c u b a te d a t 37° f o r 30 m in . The r e a c t io n m ix tu re

was in c r e a s e d i n volum e to 50 yul w ith NTE, p h en o l e x t r a c te d ,

ethanol p rec ip ita ted , washed with 955* ethanol, and ly o p h ilis e d .

U s u a lly th e DNA was th e n r e s t r i c t e d in 20 yul w ith a secon d 1

r e s t r i c t i o n e n d o n u c le a se to g iv e u n iq u e ly l a b e l l e d fr a g m e n ts ,

20 y 1 d y e s in g ly c e r o l ( 0 .0 2 5 $ w t /v o l brom ophenol b lu e ,

0 .0 2 5 $ w t /v o l x y le n e c y a n o l FP i n 1 0 $ v o l / v o l g l y c e r o l )4 , 4 4

added , and th e fra g m en ts se p a r a te d by e l e c t r o p h o r e s i s on a

4 $ p o ly a c r y la m id e g e l i n 0 .5 5 x TBE a t 20 V/cm.4

In one ex p erim en t ( s e e P ig u re 0 3 .1 6 ) , TagI s i t e s were

l a b e l l e d a t 3* t e r m in i . 2 j tg DNA c le a v e d w ith TagI was

in c u b a te d a t 1 5 ° f o r 1 hr i n 50 /* ! o f 0 .0 5 M T ris-H C l pH 7 .8 ,

0 .0 0 5 M MgCl2 » 0 .0 0 1 M d i t h i o t h r e i t o l c o n t a in in g 5 />*1

c*-^P-dC TP and 5 U o f th e Klenow fragm en t o f E . c o l i

DNA p o ly m era se I . The 3 1 l a b e l l e d TagI fra g m en ts w erei f r* rse p a r a te d by e le c t r o p h o r e s i s on an 8 $ p o ly a c r y la m id e 9*3 M

4u rea s e q u e n c in g g e l (d e sc r ib e d b e lo w ) . The r e q u ir e d fragm en t

f o r t u i t o u s l y m ig ra ted a s two s e p a r a te s tr a n d s w hich were

i s o l a t e d d i r e c t l y from th e g e l .

P o ly a c r y la m id e g e l s c o n ta in in g t e r m in a l ly - la b e l l e d

r e s t r i c t i o n fra g m en ts were a u to ra d io g ra p h ed w et w ith

r a d io a c t iv e in k m arkers, and bands e x c is e d and tr a n s fe r r e d

to 1 .5 ml Eppendorf tu b e s . The p o ly a c r y la m id e s l i c e s were

- 66 -

cru sh ed w ith s i l i c o n ! s e d g l a s s r o d s , 0 .6 ml o f 0 .1 $ w t /v o l

sodium d o d e c y l su lp h a te in NTE added to each tube and

in c u b a te d o v e r n ig h t a t 3 7 ° . The tu b e s were c e n tr ifu g e d in

an Eppendorf c e n t r i f u g e f o r 5 m in, th e su p e r n a ta n t removed

and th e p e l l e t r e - e x t r a c t e d w ith 0 .6 ml 0 .1 $ sodium d o d ec y l

s u lp h a te in NTE. The two su p e r n a ta n ts were com bined i n a

15 ml capped p l a s t i c tu b e , and e th a n o l p r e c ip i t a t e d in th e

p r e se n c e o f 30 Mg y e a s t RNA. A fte r c e n t r i f u g a t io n a t 2000

r .p .m . f o r 30 min th e r a d io a c t iv e p e l l e t s w ere r e d is s o lv e d

in 0 .2 ml 0 .1 M sodium a c e t a t e pH 7 .5 , and 0 .1 ml 1 $ w t /v o l

cetyltrim ethylam m onium bromide was added to ea ch . The

1 .5 ml Eppendorf tu b es were k ep t on i c e f o r 15 min and then

c e n t r i fu g e d a t 15000 r .p .m . f o r 15 min a t 4° in an MSE

c e n t r i f u g e . The p e l l e / t s were washed tw ic e w ith 0 .1 M

sodium a c e t a t e pH 7 .5 in 7 0 $ e th a n o l, once w ith 7 0 $ e th a n o l,

once w ith 9 5 $ e th a n o l, and th en l y o p h i l i s e d . The DNA was* T

d is s o lv e d in w ater p r io r to th e c h em ica l d e g r a d a tio n

r e a c t io n s f o r n u c le o t id e se q u e n c in g . In some ex p er im en ts

i s o l a t e d fr a g m en ts w ere la b e l l e d a t both t e r m in i and so th e y

were d ig e s t e d w ith a secon d e n d o n u c lea se a f t e r th e e th a n o l

p r e c i p i t a t i o n s t e p , and u n iq u e ly l a b e l l e d fra g m en ts were

i s o l a t e d a f t e r e le c t r o p h o r e s i s on a secon d 4 $ p o ly a c r y la m id e4

g e l . In a l l th e s t e p s d e s c r ib e d above th e w h ereab ou ts o f

th e l a b e l l e d DNA was fo llo w e d u s in g a r a d ia t io n m o n ito r .

- 67 -

D e te r m in a tio n o f th e n u c le o t id e seq u en ce o f DNA

The c h em ica l d e g r a d a tio n method was u sed (Maxam and

G ilb e r t , 1 9 8 0 ) . U n iq u ely 5 ’ - l a b e l l e d DNA was d is s o lv e d i n

25 /11 w a ter and a l iq u o t s put in to f i v e 1 .5 ml E ppendorf tu b e s

on i c e . R ea g en ts were th en added a s d e s c r ib e d below in o rd er

to m od ify gu an in e (G ), gu an in e and a d en in e (G+-A), a d en in e and

to a l e s s e r e x te n t c y t o s in e (A>C), c y t o s in e and thym ine (C+T),

o r c y t o s in e (C) r e s id u e s . A fte r n e u t r a l i s i n g th e r e a g e n t s ,

th e DNA was t r e a te d in each c a s e w ith p ip e r id in e i n o rd er to

c le a v e th e DNA stra n d a t th e p o s i t i o n s o f m o d if ie d r e s id u e s .

The t r e a te d DNA sam p les were then e l e c t r o p h o r e t i c a l l y

s e p a r a te d . For th e ch em ica l d e g r a d a tio n r e a c t io n s a l l r e a g e n ts

were i c e - c o l d , and m a n ip u la t io n s were c a r r ie d o u t s p e e d i ly

and , where a p p l ic a b le , on i c e . The w h ereab ou ts o f th e

r a d io a c t iv e DNA was fo llo w e d a t a l l s t a g e s w ith a hand

r a d ia t io n m o n ito r . R e a c tio n t im e s were e s t a b l i s h e d e m p ir ic a l ly .

1 . G r e s id u e s . 2 0 0 / i l DMS b u ffe r ( 0 .0 5 M sodium c a c o d y la te

pH 8 . 0 , 0 .0 1 M MgCl2 , 0 .0 0 1 M EDTA) and 1 /CL s o n ic a te d c a l f

thymus DNA a t 1 mg/ml were added to 5 /*1 e n d - la b e l le d DNA.

1 / i l d im e th y lsu lp h a te (DMS) was added in a fume hood to th e

tu b e , w h ich was mixed and th en in c u b a te d a t 25° f o r 2 -1 0 m in .

The tu b e was t r a n s fe r r e d to i c e and 50 /*1 DMS s to p added

( 1 .5 M sodium a c e t a t e pH 7 .0 , 1 M 2 -m e r c a p to e th a n o l, 100 /x g /m l

tRNA) and r a p id ly m ixed . 750 yul e th a n o l was added and m ixed ,

and th e tu b e p u t in an e t h a n o l- s o l id CO2 b a th f o r 5 min

( f i r s t e th a n o l p r e c i p i t a t i o n ) . The tu be was c e n t r i fu g e d f o r

5 min a t 4° in an Eppendorf 5412 c e n t r i f u g e , and th en

t r a n s f e r r e d to i c e . The su p er n a ta n t was removed w ith a

P a s te u r p i p e t t e anu th e p e l l e t d i s s o lv e d i n 250 /CL 0 .3 M

sodium a c e t a t e pH 6 .5 c o n ta in in g 40 / ig/m l y e a s t RNA.

- 68 -

750 yul e th a n o l was add ed , and th e tu b e m ixed and p u t f o r 5 min

i n an e t h a n o l - s o l id CO2 b ath (seco n d e th a n o l p r e c i p i t a t i o n ) .

The su p e r n a ta n t was removed a f t e r 5 min c e n t r i f u g a t io n , and

750 yul e th a n o l c a r e f u l l y added to th e p e l l e t . The tu b e was

c e n t r i fu g e d f o r 2 min a t 4° and th e su p e r n a ta n t d is c a r d e d .

The p e l l e t was d r ie d in a l y o p h i l i s e r . 100 /□ . 1 M p ip e r id in e

was added and th e tu b e h ea ted a t 9 0 ° f o r 30 m in . The s o lu t io n

was fr o z e n on s o l i d CO2 and l y o p h i l i s e d u n t i l no i c e

rem ain ed . 1 0 / i l w a ter was added to th e d r ie d p e l l e t , m ixed ,

f r o z e n and l y o p h i l i s e d , and th en t h i s s te p was r e p e a te d . The

f i n a l p e l l e t was d is s o lv e d in 5 -2 0 yul form am id e-d yes

(8 0 $ v o l / v o l form am ide, 0*55 x TBE, 0 .1 $ w t /v o l x y le n e c y a n o l4 ^ 4

FF, 0 .1 $ w t /v o l brom ophenol b lu e ) .4

2 . G+A r e s id u e s . 17 /»1 w a te r , 1 yul s o n ic a te d c a l f thymus

DNA a t 1 m g/m l, and 2 ^ 1 M p y r id in iu m fo rm a te pH 2 were

added to 3 / I e n d - la b e l le d DNA. The tu b e was in c u b a te d a t

2 5 ° f o r 3 0 -1 2 0 m in, and th en fr o z e n and l y o p h i l i s e d . The

p e l l e t was d is s o lv e d i n 20 yul w a ter , fr o z e n th en l y o p h i l i s e d .

Treatm ent w ith p ip e r id in e and su b seq u en t s t e p s were u n d ertak en

a s f o r th e G r e a c t io n .

3 . A>C r e s id u e s . 1 0 0 yd. 1 .2 M NaOH, 0 .0 0 1 M EDTA and 1 / I

s o n ic a te d c a l f thymus DNA a t 1 mg/ml were added to 5 / L en d -

l a b e l l e d DNA, and in c u b a te d a t 90° f o r 2 -1 0 m in. 150 yul

1 i.i a c e t i c a c id , 1 yul y e a s t KNA a t 10 m g/m l, and 750 ywl

e th a n o l were added . A fte r th e norm al e th a n o l p r e c i p i t a t i o n

p r o c ed u r e , tr ea tm en t w ith p ip e r id in e and su b seq u en t s t e p s were

c a r r ie d o u t a s f o r th e G r e a c t io n .

4 . C-KF r e s id u e s . 15 / - I w a ter , 1 yul s o n ic a te d c a l f thymus

DNA a t 1 m g/ml, and 30 / - l h y d ra z in e were added w ith m ix in g

to 5 /* ! e n d - la b e l le d DNA, and in cu b a ted a t 25° f o r 2 -1 0 m in .

200 jx l h y d r a z in e s to p ( 0 ,3 M sodium a c e t a t e pH 6 .5 , 0 .0 0 0 1 M

EDTA, 50 jug/m l tRNA) was th en added and th e tu b e m ixed.

750 jjOl e th a n o l was added and th e f i r s t e th a n o l p r e c i p i t a t i o n

c a r r ie d o u t . The secon d e th a n o l p r e c i p i t a t i o n and su b seq u en t

s t e p s were done a s f o r th e G r e a c t io n .

5 . C r e s id u e s . The s t e p s f o r th e C+T r e a c t io n were c a r r ie d

o u t e x c e p t t h a t th e 15 >ul w a ter in th e h y d ra z in e r e a c t io n

was r e p la c e d by 15 /d. 5 M NaCI.

A liq u o ts o f 3 -5 1 o f r e a c te d la b e l l e d DNA d is s o lv e d

in form am id e-d yes were h ea ted a t 1 0 0 ° f o r 2 min and quenched

on i c e , b e fo r e lo a d in g on to se q u e n c in g g e l s . For g e l

p r e p a r a t io n , s to c k s o lu t io n s o f d e io n is e d 2 0 # w t /v o l

a cry la ra id e (2 9 :1 w t:w t a c r y la m id e :N N '-m e th y le n e b isa c r y la m id e )

in 8 M u r e a , 10 x TBE, 10 M u r e a , and 1 0 # w t /v o l ammonium4 ^

p e r s u lp h a te were u s e d . S o lu t io n s o f th e a p p r o p r ia te

a c r y la m id e c o n c e n tr a t io n were p rep ared by d i l u t i n g th e 2 0 #

a c r y la m id e s o lu t io n w ith 10 M u r e a . 10 x TBE and 1 0 #4

ammonium p e r s u lp h a te were added to f i n a l c o n c e n tr a t io n s o f

0 .5 5 x TBE and 0 .0 6 7 # , r e s p e c t i v e l y , and th e p o ly m e r is in g4

c a ta ly s t TEMED added to 0 .0 0 0 5 # v o l /v o l . Sequencing g e ls

o f dimensions 450 x 230 x. 0 .5 mm were poured, using 60 ml

so lu tio n for each g e l . Three concentrations o f g e l were

used: 6# polyacrylam ide, 9*3 M urea; 8# p61yacrylamide,4 4

9 .3 M u r e a ; 1 9 # p o ly a c r y la m id e , 8 M u r e a . P r e - e le c t r o p h o r e s is4

o f g e l s was c a r r ie d o u t f o r 1 - 1 .5 h r a t 40 W ( 1 .5 - 2 kV)

b e fo r e lo a d in g th e sa m p les . G els were s u b je c te d to

e l e c t r o p h o r e s i s a t 40 W such th a t 1 9 # p o ly a c r y la m id e r e s o lv e d

1 -3 0 n (brom ophenol b lu e marker 15 cm down g e l ) , 0 #

p o ly a c r y la m id e r e s o lv e d 2 0 -8 0 n (x y le n e c y a n o l marker 15 cm

down g e l ) and 8 0 -1 2 0 n (x y le n e cy a n o l marker to bottom o f g e l ) ,

- 7 0 -

and 6?o p o ly a c r y la m id e r e s o lv e d 1 2 0 -1 5 0 n (x y le n e c y a n o l to

bottom o f g e l ) and 150 n and la r g e r (se co n d x y le n e c y a n o l

marker a p p lie d when th e f i r s t had rea ch ed th e bottom o f th e

g e l , th en a llo w e d to m ig r a te 15 cm down g e l ) .

A fte r e le c tr o p h o r e s is one g la s s p la t e was removed.

The g e l was wrapped in a p la in S t e r i l i n bag and a u to ra d io g ra p h ed

f o r 1 -3 0 d ays a t -7 0 ° u s in g p r e f la s h e d Kodak X-Omat H f i lm*

and a Dupont Cronex p h o sp h o tu n g sta te s c r e e n .

Table .Bl.l

Source b a c te r ia fo r r e s t r i c t i o n en d on u cleases

used in t h i s s tu d y , w ith growth tem peratures

fo r batch l iq u id c u l t u r e s .

Those b a o te r ia n o t a ss ig n e d a growth

tem perature were su p p lie d a s fr o z e n c e l l

p a s te s by th e M ic r o b io lo g ic a l R esearch

E sta b lish m e n t, P orton Down, W ilt s h ir e , and

by Dr J . Arrand.

-p

c~\

O I

0) Xt

Table B1.2

Elution of restrlotlon endonucleases from ion exchange columns*The molarity of TCC1 In the first ooluisn fraotlon containing restriction activity was calculated from the linear KC1 gradient used for elution*

Table B1.2

restrictionendonuclease

approx* eluting molarities (KCl)DE52 Biorex

Xba I 0.2 _ _

Hind III 0 0.3BcoR I 0.13 0.45Bgl II 0.18 0.3Hna I 0.15 0.2Kpn I Q.1 0.15BamH I 0.1 0.3Pvu II 0.15 0.4

Table B1.5

N om en clatu re o f recom b in an t p la sm id s b ea r in g HSV-1 (pGX)

o r HSV-2 (pGZ) DNA i n s e r t s . T r iv ia l names a re u sed in

th e t e x t .

The i n s e r t in pGZ12 c o n s i s t s o f HSV-2 H in d III £ and

a b o u t 1 x 10 d a l to n s o f a d ja c e n t seq u en ce from

HSV-2 H in d l l l 1 ( s e e P ig u re C 2 .4 ) . I t may have

o r ig in a t e d from a d e f e c t i v e DNA m o le c u le .

c lo n e dfragm en t

t r i v i a lname

sta n d a rdname

HSV-2 H in d III a a pGZ26

HSV-2 H in d III to to pG Zll

HSV-2 H in d iI I e e pGZ25

HSV-2 H in d iI I h h pGZIO

HSV-2 H in d III 1 1 pGZ23

HSV-2 H in d iI I n n pGZ32

HSV-2 H in d iI I o 0 pGZ34

HSV-2 H in d III 12 12 pGZ12

HSV-2 BarnHI g £ pGZ6

HSV-2 BamHI g 1 pGZl

HSV-1 BamHI £ I pGXl

HSV-1 BamHI k k l pGX2

HSV-1 BamHI k k2 pGX7

HSV-1 BamHI k . & PGX5

HSV-1 BamHI k k l PGX4

HSV-1 BamHI I ' g2 pGX8

HSV-1 BamHI k ( s t r a i n USA-8)

i pGXIO

Table 3 1 .4

S i t e s f o r r e s t r i c t i o n e n d o n u c le a se s u sed in t h i s

Work (from R o b e r ts , 1 9 8 1 ) . C leavage p o s i t i o n s are shown

by a p o s tr o p h e s .

T a b l e B l . 4

A lu l AGr* CT Kind I I I A1 AGCTT

A val C'ByCGPuG H ln f l G1 AN TC

A v a il G* GA/TCC Hpal GTT1 AAC

Bam lil G» GATCC Kpnl GGTAC’ C

B e l l T’ GATCA MboII G AA GAN u N NN N NN1

B ^ l l GCCKKNN'NGGC M nll CCTC (5 -•10 n . ~ 3 ' )

B ^ lI I A’ GATC'if P vu II CAG'CTG

BstN I CC1 A/TOC Sac I GAGO T1C

C la l AT'CGAT Sau3AI 1GATCf

D del C’ TNAG S s t I GAGCT'C (a s S a o l)

gcoKI G! AATTC S s t l l CCGC1 GG

l l a e l l PuCCGC'Py Sraal CCC1GGG

lia o I I I GG'CC Tag I T»CGA

n h a l GCG’C Xbal T’CTAGA

H ln cII GTPyPuAC Xhol C * TC GAG

P A & T C

it i!i S U L i1 S

A N D

D I S C U S S I O N

SECTION 1

liAPPIHG OP RESTRICTION ENDONUCLEASE SITES

ON HSV-1 AND HSV-2 DNA

- 71 -

INSULTS


R e s t r ic t io n maps f o r th e genom es o f HSV-1 s t r a in 17

and HSV-2 s t r a in HG52 which were a v a i la b le a t th e

commencement o f t h i s work were f o r X b al, H i n d l l l , BcoRI,

B g l l l and Hpal , and a l s o Kpnl f o r s t r a i n HG52 (W ilk ie , 1976;

C o r t in i and W ilk ie , 1 9 7 8 ) . These e n d o n u c le a se s produce

r e l a t i v e l y few r e s t r i c t i o n fra g m en ts from HSV DNA, and so

.the o b j e c t o f th e work d e s c r ib e d h e re was t o map HSV-1 and

HSV-2 DNA f o r e n d o n u c le a se s w hich produce many fra g m en ts , in

o rd er to be a b le to d e f in e more c l o s e l y th o se s t r u c t u r a l

and f u n c t io n a l a s p e c t s o f th e genome u nder i n v e s t i g a t i o n .

M o lecu la r w e ig h t c a l ib r a t io n

The s ta n d a r d s u sed to d eterm in e th e m o le c u la r w e ig h ts

o f r e s t r i c t i o n fra g m en ts were th e EcoRI d i g e s t o f lambda

DNA (Thomas and D a v is , 1 9 7 5 ) , th e H in d ll d i g e s t o f jrfx 174

nJMA (Mania i i s e t a l . , 1 9 7 5 ) , and th e Hind I I d i g e s t o f lambda

DNA (M a n ia tis e t a l . , 1 9 7 5 ) . These d i g e s t s were u sed to£

c a l ib r a t e m o le c u la r w e ig h ts i n th e r a n g e s o f 1 -1 0 x 106 6

0 .1 - 1 x 1 0 , and 0 .0 7 - 0 .1 x 10 , r e s p e c t i v e l y .

F ig u re C l . l shows th e g e l u sed to c a l ib r a t e

th e la r g e r fra g m en ts o f th e Kpnl , BamHI, X h o l, and Hpal

d i g e s t s o f HSV-1 DNA. The la r g e r fra g m en ts o f HSV-1 P vuII

were c a l ib r a t e d u s in g HSV-1 Hpal fragm en t m o le c u la r w e ig h ts

(W ilk ie , 1 9 7 6 ) . HSV-2 B g ll l fragm en t m o le c u la r w e ig h ts

(C o r t in i and W ilk ie , 1978) were u sed to c a l i b r a t e la r g e r

HSV-2 BamHI fr a g m e n ts .

F ig u re C l .2 shows th e a u to ra d io g ra p h u sed to c a l ib r a t e

th e sm a lle r fra g m en ts o f HSV-1 BamHI , HSV-1 X h o l, and HSV-2

BamHI. Com parison o f HSV-1 Xhol w ith HSV-1 P v u II a llo w ed

- 72 -

m o le c u la r w e ig h ts o f th e s m a lle r P vu II fra g m en ts to be

d e te rm in ed , HSV-1 Kpnl b 1 and c 1 were c a l ib r a t e d a g a in s t

th e HSV-1 BamHI d i g e s t , and HSV-I BamHI k '-n * a g a in s t lambda

H indl l fra g m en ts on a p o ly a c r y la m id e g e l .

The d is t a n c e m ig ra ted by s ta n d a rd bands was p lo t t e d

a g a in s t th e lo g a r ith m o f th e m o le c u la r w e ig h t , and t h i s cu rv e

was u sed to deduce m o le c u la r w e ig h ts o f r e s t r i c t i o n fra g m en ts

(Tables C 1.1-C 1.5). I t was assumed that the standard curve

was v a l i d f o r a l l r e s t r i c t i o n fr a g m e n ts; h ow ever, th e

n u c le o t id e c o m p o s it io n o f DNA m ight a f f e c t i t s e le c t r o p h o r e t ic*

m o b il i t y and h en ce th e a p p a ren t m o le c u la r w e ig h ts o f some

fr a g m e n ts .

R e la t iv e m o la r i t i e s o f r e s t r i c t i o n fra g m en ts

F ig u re C l . 3 show3 r e s t r i c t i o n p r o f i l e s o f

in v i v o ^ P - l a b e l l e d HSV-1 and HSV-2 DNA. A u to d en sito m etry

o f t h e s e , and o th e r , a u to r a d io g r a p h s was c a r r ie d o u t to

d e term in e th e r e l a t i v e m o la r i t i e s o f bands in th e p r o f i l e s

(T a b le s C 1 .1 -C 1 .5 ) . P o t e n t ia l s o u r c e s o f e r r o r in such

e s t im a t io n s in c lu d e in a c c u r a te m o le c u la r w e ig h ts , d e g r a d a tio n

o f DNA, a n o n - l in e a r r e sp o n se o f f i lm to r a d ia t io n ex p o su re ,

and fra g m en t h e t e r o g e n e i ty ( f o r in s t a n c e , a p r o p o r t io n o f

th e HSV-1 BamHI j o in t fragm en t k c o n ta in s two a seq u e n c es

in s t e a d o f o n e , and t h e r e fo r e co m ig ra ted w ith BamHI .

N e v e r t h e le s s , in g e n e r a l th e m easured r e l a t i v e m o la r i t i e s

a re in agreem en t w ith th o se p r e d ic te d from th e p h y s ic a l maps

( b e lo w ) . Bach o f th e r e s t r i c t i o n e n d o n u c le a se s f o r which

HSV-1 o r HSV-2 DNA was mapped in t h i s work h as a s i t e i n one

or b o th o f th e s e t s o f in v e r te d r e p e t i t i o n s and th e r e fo r e

p ro d u ces no q u a r ter -m o la r fr a g m e n ts , s in c e o n ly e n d o n u c le a se s

w ith no s i t e s in r e p e t i t i v e se q u e n c e s show t h i s c h a r a c t e r i s t i c .

- 73 -HSV-1 Kpnl p h y s ic a l map: r e c le a v a g e d a ta

F ig u r e s C 1 .4 -C 1 .5 show th e p r o d u c ts o f Kpnl c le a v a g e

o f i s o l a t e d HSV-1 r e s t r i c t i o n fra g m en ts o f known map

p o s i t i o n s . F ig u re C l . 6 shows a summary o f th e s e d a ta .

In th e f o l lo w in g argum ent m o le c u la r w e ig h ts a re g iv e n in m i l l i o n s .

1 . The S seg m en t.

.Both Hind i I I g and B g l l l 1 gave K£nl k a s an S te r m in a l

fra g m en t, and BcoRI h p r o d u c ts show Kpnl h to map in Us-

C lea v a g e o f KcoKI h and k in d ic a t e a th a t S c o n ta in s th r e e

fr a g m e n ts . The t h ir d Kpnl fragm en t i s la r g e r than

H indl l l m ( 4 . 6 ) , s in c e t h i s fragm en t was n o t c le a v e d oy

K pnl, and th e p r o d u c ts o f H indI I I n s u g g e s t th a t th e m o le cu la r

w e ig h t o f th e th ir d fragm en t i s 5*5 ( 4 .6 + 0 . 9 ) . S im i la r ly ,

c le a v a g e o f BcoRI h and k im p l ie s a s i z e o f 5 .4 ( 3 .5 + 1 .9 )

f o r th e t h ir d fra g m en t. T h is s i z e c o r r e sp o n d s to th a t o f

Kpnl j , w hich in d eed was a p ro d u ct o f th e c le a v a g e o f

Xbal a , X bal be, Hpal c d e , and JBglll g h . B oth Kpnl j. and k

w ere h a l f m olar in th e HSV-1 Kpnl d ig e s t (T a b le C l . l ) .

T h e re fo r e th e o rd er o f Kpnl fra g m en ts in S i s j. h k .

T h is c o n c lu s io n was u sed to deduce w h ich p r o d u c ts o f

Hpal gh were produced froip Hpal g and w hich from h , and th e

p r o d u c ts a re shown s e p a r a te ly in F ig u re C l. 6,. S im ila r

r e a s o n in g was a p p lie d w ith Hpal c d e , B g ll l g h , and X bal be

to d eterm in e th e c le a v a g e p r o d u c ts o f in d iv id u a l fr a g m e n ts .

2 . THL/IH L.

Fragm ents c o n ta in in g th e 1 term in u s gave Kpnl r a s a

common p r o d u c t . T h ere fo re Kpnl r maps a t th e 1 te r m in u s .

T n is a l lo w s th e p r o d u c ts o f H in d l l l h and i to be

deduced from H in d l l l h i .

- '74 -

3 . J o in t fr a g m e n ts .

The s i z e s o f L and S te r m in a l fr a g m e n ts in d ic a t e t h a t

th e two h a l f m olar j o i n t fra g m en ts have m o le c u la r w e ig h ts o f

'7.9 ( 5 .5 + 2 .4 ) and 7 .2 ( 4 .8 + 2 . 4 ) . Hpal a gave Kpnl a ( 8 . 0 ) ,

and Kpnl e (7 * 0 ) was a p rod u ct o f r e le v a n t c le a v a g e s ( Hpal

c d e . B g ll l e , Xbal q , Xbal b e ) • The j o in t fra g m en ts a r e

t h e r e fo r e a and e .

4 . J u n c t io n s o f TRj/ I R j, w ith Uj,.

There i s only"one" Kpnl s i t e i n TR j/IR ^, s in c e o n ly one

Kpnl fragm en t was common to b o th 1 te r m in i ( Kpnl r ) .

T h ere fo re fra g m en ts a d ja c e n t to t h i s s i t e m ust ex ten d in t o

UL, and h en ce must be g r e a te r in s i z e th an 3 .6 ( 6 . 0 - 2 . 4 ) .

The o n ly fragm en t from B g ll l f and Xbal d w h ich m eets t h i s

req u irem en t i s Kpnl g . S im i la r ly , th e r e s u l t s from B g l l l q

and k g iv e a s i z e o f 8 .1 ( 7 .0 + 1 .1 ) f o r th e o th e r ju n c t io n

fra g m en t, and t h i s c o rresp o n d s to Kpnl b . T h ere fo re th e

fra g m en ts so f a r deduced a r e , a s sp aced a lo n g th e genom e,

r b . . . . g r / j h k .

5 . Ujj from l e f t to r i g h t .

R e c lea v a g e o f H indl l l h and B g l l l k show s t h a t K gnl x

i s a d ja c e n t to Kpnl b . C leavage o f B g l l l o and k , b e a r in g

in mind t h a t Hpal k and u were n o t c le a v e d by K pnl, in d ic a t e s

th e s i z e o f th e n e x t fragm en t i s 6 .9 ( 4 . 6 + 2 . 3 ) - T h is

co rresp o n d s to Kpnl f , w hich was produced from Xba I c .

Comx)arison o f B g l l l p and Hpal j, c le a v a g e show s th a t th e n e x t

two fra g m en ts a re Kpnl q then w. In l i k e manner th e p r o d u c ts

o f Xbal c , B g l l l m and Hpal i d em on stra te t h a t th e n e x t th r e e

fra g m en ts a r e Kpnl i , b ’ th en m. T h ere fo re th e o rd er so f a r i s

r b y f q w i b ’ m . . . . g r / q h k .

- 75 -

The n e x t r e g io n c o n ta in s Kpnl n , p , t , v , x and a 1 ,

a c c o r d in g to c le a v a g e o f X bal f . The fragm en t a t th e l e f t

hand end o f t h i s group i s shown by c le a v a g e o f Hpal b to be

Kpnl t . P r o d u c ts o f B g ll l i in d ic a t e t h a t Kpnl n and p a re

th e n e x t two fr a g m e n ts , but t h e s e ca n n o t be ord ered w ith

r e s p e c t to ea ch o th e r by r e c le a v a g e m ethods b ecau se none o f

th e o th e r e n d o n u c le a se s c l e a v e s w ith in them . The n e x t fragm ent

i s l a r g e r in s i z e t h a t 1 * 0 , and c le a v a g e o f EcoRI m th e r e fo r e

show s th e o rd er o f Kpnl v , x , §t' i s y a* x . So th e o r d e r o f

fra g m en ts w ith in Xbal f i s Kpnl t (nq) v a* x .

The n e x t fragm en t i s la r g e r in m o le c u la r w e ig h t than •

6 .0 , a s shown by com p arison o f th e p r o d u c ts o f Hpal h and

EcoRI 1 . Kpnl c , a p ro d u ct o f B g l l l d , i s th e o n ly fragm ent

m eetin g t h i s r e q u ir e m e n t. C lea v a g e o f Xbal e , H in d l l l k ,

EcoRI a , B g l l l d and Hpal f show t h a t th e n e x t th r e e

fr a g m en ts a re Kpnl 1 c* s , and com parison o f th e B g l l l g ,, - . . * . . . . .

Hpal e and EcoRI i r e s u l t s d em o n stra te t h a t th e s e a re

fo l lo w e d by Kpnl d z u . The f i n a l fra g m e n t, from c le a v a g e

o f B g l l l f and Xbal d , i s Kpnl p .

In c o n c lu s io n , th e o rd er o f Kpnl fr a g m en ts w ith in

th e HSV-1 genome i s

r b y f q w i b ' m t (nq) v a 1 x c 1 c 1 s d z u o g r / p h k ,

w ith Kpnl a and e r e s u l t i n g from f u s io n a t th e j o i n t o f

Kpnl p and r , and k and r , r e s p e c t i v e l y . T h is c o n c lu s io n

a c c o u n ts f o r a l l th e bands in th e HSV-1 Kpnl r e s t r i c t i o n

p r o f i l e in t h e ir r e s p e c t iv e m o la r i t i e s . No fragm en t s m a lle r

than Kpnl c 1 h as been d e t e c t e d .

P ig u re C l . 6 , show ing a summary o f c le a v a g e d a ta ,

g iv e s th e p r o d u c ts o f in d iv id u a l fra g m en ts w ith in c o m ig r a tin g

bands a s a lr e a d y d e s c r ib e d , in c lu d in g Hpal o p q r .

- 76 -HSV-1 Kpnl p h y s ic a l map: d ou b le d i g e s t d a ta

P ig u re C l . 7 shows th e r e s u l t s o f s i n g l e and d o u b le

d ig e s t io n o f HSV-1 BNA. P ig u r e s C l . 8 - C l . 9 . show th e

r e s u l t s o f c le a v in g i s o l a t e d Kpnl fra g m en ts w ith o th e r

e n d o n u c le a s e s . These d a ta a re sum m arised in T ab le C l . 6 .

The r e s u l t s a re c o n s i s t e n t w ith th e p h y s ic a l map deduced

from r e c le a v a g e d a ta .

HSV-1 Kpnl p h y s ic a l map: h y b r id is a t io n d a ta

H y b r id is a t io n d a ta a r e p ro v id ed by th e c r o s s - b l o t

shown i n P ig u r e C l ,1 0 . Each s p o t on th e a u to r a d io g ra p h

i n d i c a t e s a r e g io n o f seq u en ce hom ology betw een th e two

fra g m en ts co n c er n e d . Por exam p le , Kpnl wx h y b r id is e d to

Hpal b and q , and r e c le a v a g e d a ta show t h a t Kpnl w maps

in Hpal q and Kpnl x i n Hpal b . The d a ta a re sum m arised in

P ig u re C l .1 1 , u s in g th e p h y s ic a l map a lr e a d y d ed u ced , w ith

w hich th e y a r e c o n s i s t e n t . Homology betw een th e 1 and S

te r m in i i s e x p la in e d by th e p r e se n c e a t both te r m in i o f

th e te r m in a l r e p e t i t i o n , or a seq u e n c e , w hich i s a l s o

p r e s e n t a t th e j o in t o f th e L and S se g m e n ts .

HSV-1 Kpnl p h y s ic a l mapt r e l a t i v e o rd er o f fra g m en ts n and p

None o f th e p r e v io u s m ethods a llo w e d th e o r d e r in g o f

Kpnl n and q , a s no r e le v a n t r e s t r i c t i o n s i t e i s p r e s e n t

w ith in th e r e g io n c o n ta in in g them . Work d e s c r ib e d i n S e c t io n

2 show s th a t th e genom es o f HSV-1 and HSV-2 a r e g e n e r a l ly

c o l in e a r , and s t u d ie s o f in t e r t y p ic reco m b in a n ts i s in

a cco rd w ith t h i s h y p o th e s is ( e . g . P r e s to n e t a l . , 1 9 7 8 ) .

T h erefo re a r e s t r i c t i o n s i t e w ith in HSV-2 seq u e n c es hom ologous

to HSV-1 Kpnl n and q co u ld be used to o r ie n t a t e th e s e two

fr a g m e n ts . The r e s t r i c t i o n s i t e u sed was th e B g ll l s i t e

betw een HSV-2 B g ll l q and o (C o r t in i and W ilk ie , 1 9 7 8 ) .

- 77 -

In P ig u re C l .12 th e r e s u l t i s shown o f h y b r id is in g

Kpnl no and q to n i t r o c e l l u l o s e b lo t s t r i p s c o n ta in in g

HSV-1 Hpal or HSV-2 B g ll l r e s t r i c t i o n fr a g m e n ts . Kpnl no

h y b r id is e d to HSV-1 Hpal b, e and q . P resum ably th e form er

was ow ing to h y b r id is a t io n o f Kpnl n and th e l a t t e r two to

Kpnl o . H y b r id is a t io n to Hpal i was p ro b a b ly due to

c o n ta m in a tio n o f Kpnl nq by Kpnl m. Kpnl q h y b r id is e d to

Hpal b o n ly , show ing th a t t h i s fra g m en t was r e l a t i v e l y p u re .

With HSV-2 B g ll l b l o t s t r i p s Kpnl no h y b r id is e d to B g ll l i

'and but n o t o . H y b r id is a t io n to B g ll l i was presum ably

due to Kpnl o and to B g ll l j. by Kpnl n , a s th e y are

d i f f e r e n t i a t e d by p h y s ic a l map p o s i t i o n . Kpnl q h y b r id is e d

to B g l l l q and o . These r e s u l t s s u g g e s t t h a t Kpnl n and q

a re in th e o rd er n q in th e HSV-1 genom e.

HSV-1 Kpnl p h y s ic a l map

The p h y s ic a l map c o n s i s t e n t w ith a l l th e d a ta i s

shown in P ig u re C l .1 8 .

P h y s ic a l maps f o r o th e r r e s t r i c t i o n e n d o n u c le a se s

The d e r iv a t io n o f th e HSV-1 Kpnl p h y s ic a l map h a s been

d e s c r ib e d , and maps o f HSV-1 DNA f o r BamHI . PvuI I and X h o l,

and o f HSV-2 DNA f o r BamHI were c o n s tr u c te d in s im i la r m anner.

A u torad iograp h s o f r e c le a v a g e , d o u b le d ig e s t io n and

h y b r id is a t io n ex p er im en ts have n o t been in c lu d e d , but in s t e a d

a re summarised in d iagram m atic (F ig u r e s C 1 .1 3 -C 1 .1 7 ) o r

ta b u la r form (T a b le s C 1 .7 -C 1 .1 2 ) . P h y s ic a l maps deduced

from r e c le a v a g e d a ta were con firm ed by d o u b le d i g e s t i o n and

h y b r id is a t io n e x p e r im e n ts , e x c e p t f o r HSV-1 P v u II , f o r w hich

h y b r id is a t io n ex p er im en ts were n o t c a r r ie d o u t . A ll

d e t e c ta b le HSV-1 Kpnl and BamHI fra g m en ts were mapped, but

in th e o th e r d i g e s t s an undeterm ined number o f v e ry sm a ll

- 78 -

fra g m en ts were n o t r e s o lv e d and t h e r e f o r e rem ained unmapped,

P in a l r e s t r i c t i o n maps a r e shown in P ig u r e s C 1.18 and C l .1 9 ,

and a l ig n e d HSV-1 and HSV-2 maps in P ig u r e C l .2 0 .

The m ethods d e s c r ib e d above f a i l e d to unam biguously

map a few fra g m en ts w hich were su b se q u e n tly a s s ig n e d a s f o l l o w s .

1 . K ec lea v a g e ex p e r im en ts showed th a t HSV-1 BamHI 1* and n f

a re lo c a t e d a d ja c e n t to BamHI z . The i n t e r t y p i c recom b in ant

HE4 (P ig u re 04*26) p o s s e s s e s a c r o s s o v e r in HSV-1 BamHI z ,

and produced BamHI n' but n o t l f , and t h e r e fo r e th e o rd er

HSV-1 BamHI n v z 1 ! i s th e m o s t ' ' l ik e ly .

2 . HSV-2 BamHI i and i 1 were ord ered by in f e r e n c e from d a ta

co n c er n in g th e i n t e r t y p i c recom b in ant RE4 (P ig u r e C 4 .2 6 ) .

T h is recom b in an t p o s s e s s e s an in s e r t i o n o f th e HSV-2 seq u en ce

sp an n in g th e HSV-2 BamHI i - r r e g io n in a n o th e r r e g io n o f th e

genome, a s d e s c r ib e d in S e c t io n 4 . The in s e r t e d p ie c e o f DNA

d o es n o t in c lu d e HSV-2 BamHI i 1, and th e r e fo r e i t i s l i k e l y

th a t i and r a re c o n t ig u o u s .

3 . I n t e r t y p ic reco m b in a n ts w ith a c r o s s o v e r in HSV-1 BamHI y

a s shown below la c x e d HSV-2 BamHI g ' , but produced a

c h a r a c t e r i s t i c a l l y s m a lle r fragm en t and HSV-2 BamHI m*

( e . g . HE4 s u b c lo n e s 1 , 5 , 7 , 1 5 , 1 6 , 17 in P ig u r e C 4 .2 2 ) .

These r e s u l t s s u g g e s t t h a t g ’ i s a d ja c e n t to u in th e HSV-2

BamHI map.HSV-1 DNA

x yL

— —T—— ——————1•m' q4

H i'Kg

T TTa 1 m1 g

•.

tu

HSV-1 BamHI s i t e s

HSV-2 BamHI s i t e s

HSV-2 DNA

- 79 -

Discussion

The g e n e r a l arrangem ent o f th e HSV genome h as been

a d e q u a te ly d em on stra ted and th e r e fo r e was assum ed in

d ed u c in g th e r e s t r i c t i o n maps p r e se n te d h e r e . The p r e se n c e

o f TRj/ I R jq and TRg/IRg was co n fir m ed . BamHI i s th e o n ly

r e s t r i c t i o n e n d o n u c le a se , f o r w hich th e e n t i r e HSV-2 genome

h a s been mapped, w hich c le a v e s w ith in b o th s e t s o f r e p e a t s ,

th u s d e m o n str a tin g t h e i r p r e s e n c e .

The s u b se q u e n tly p u b lish e d Kpnl and BamHI maps o f

HSV-1 s t r a i n s P and J u s t in (L ocker and P r e n k e l, 1 9 7 9 -b )d iffe r

from th o s e o f HSV-1 s t r a in 17 o n ly in th e p r e se n c e o r a b sen c e

o f a few r e s t r i c t i o n s i t e s , but in g e n e r a l a l l th r e e s t r a i n s

nave v e r y s im i la r r e s t r i c t i o n m aps. In c o n t r a s t , few i f

any r e s t r i c t i o n s i t e s a re i n e q u iv a le n t p o s i t i o n s in HSV-1

and HSV-2 DMA, even i n th e c a s e o f BamHI w h ich has many

more s i t e s in HSV DNA than p r e v io u s ly mapped e n d o n u c le a s e s .

T h is i s t r u e even o f th e more hom ologous r e g io n s betw een

th e two genom es ( s e e S e c t io n 2 o f R e s u l t s ) . Such a la r g e

d i f f e r e n c e i s o f ad v a n ta g e in d e te r m in in g th e genome

s t r u c t u r e s o f HSV-l/HSV-2 in t e r t y p ic reco m b in a n ts by

r e s t r i c t i o n en d o n u c le a se a n a l y s i s . I t ca n n o t be assum ed

th a t any r e s t r i c t i o n s i t e s in th e one genome a r e in

e q u iv a le n t p o s i t i o n s in th e o th e r , and t h e r e f o r e

r e s t r i c t i o n maps o f com parable r e g io n s o f th e two genom es

m ust be d eterm in ed s e p a r a te ly in ea ch c a s e .

S e v e r a l l i n e s o f e v id e n c e s u g g e s t t h a t th e KSV-1 and

HSV-2 genom es a re c o l in e a r , a s d is c u s s e d in S e c t io n 2 .

T h is v/as assum ed to a p p ly to th e r e g io n o f c o n ta in in g

HSV-1 Kpnl n and £ in d e te r m in in g t h e i r r e l a t i v e o r d e r

by h y b r id is a t io n to HSV-2 r e s t r i c t i o n fra g m en ts (P ig u r e

C l . 1 2 ) . I t i s p o s s ib le t h a t c o l i n e a r i t y d o es n o t a p p ly

- 80 -

in t h i s r e g io n , a lth o u g h th e s im p le s t in t e r p r e t a t io n o f

th e recom b in an t genome a n a ly s e s d e s c r ib e d by C hartrand e t a l .

(1979 and 1 980) i s t h a t c o l i n e a r i t y d o e s a p p ly .

R e s t r i c t io n fra g m en ts o r ig in a t in g from th e j o i n t

and L term in u s o f both HSV-1 and HSV-2 ENA d em o n stra ted an

a d d i t io n a l d i s c r e t e but f a i n t band m ig r a t in g a t a p o s i t i o n

3 0 0 -4 0 0 bp la r g e r th an th e m ajor s p e c ie s ( e . g . HSV-1 BamHI

k above BamHI k in P ig u re C1.3> and a f a i n t band above

HSV-1 Hpal m in P ig u re C l .7 tr a c k 1 1 ) . In some a u to r a d io g r a p h s

v e r y f a i n t bands r e p r e s e n t in g fu r th e r d i s c r e t e a d d i t io n s w ere

o b se r v e d . Pragm ents from th e S term in u s were p r e s e n t a t

t h i s r e s o lu t io n a s a s in g l e s i z e c l a s s . T hese o b s e r v a t io n s

co n firm th o s e o f Wagner and Summers (1 9 7 8 ) f o r HSV-1 and

W ilk ie e t a l . (1 9 7 7 a )fo r HSV-2. Wagner and Summers (1 9 7 8 )

co n c lu d ed t h a t th e a d d i t io n a l DNA c o m p r ise s th e a seq u en ce

and p erh ap s p a r t o f th e b s e q u e n c e . I t c o n t a in s , in f a c t ,

p r e c i s e l y th e a seq u en ce ( S e c t io n 3 o f R e s u l t s ) .

The m .w t. o f th e HSV genome can be c a lc u la t e d by

summation o f m .w t s .o f th e in d iv id u a l r e s t r i c t i o n fr a g m e n ts .

The mean m .w t. o f HSV-1 DNA, from th e fo u r d i g e s t s d e a l t w ith

h e r e , i s 9 6 . 4 ± 2 . 5 x 10 . B ea r in g in mind th e p r e se n c e o f

an unknown number o f sm a ll unmapped r e s t r i c t i o n fra g m en ts in

th e Xhol and P v u II d i g e s t s w hich have n o t been ta k en in to6 6

a c c o u n t , th e Kpnl (9 6 .7 x 10 ) and BamHI (9 7 * 4 x 10 ) m .w ts .

g iv e th e m ost r e l i a b l e e s t im a t e s . T hese s i z e s a r e i n a cco rd

w ith t h a t o f 98 x 10^ r e p o r te d by W ilk ie ( 1 9 7 6 ) . The m .w t.a ■

o f HSV-2 DNA, from th e BamHI d i g e s t , i s 97*6 x 10 > w hich- - c.

compares w ell with the s iz e o f 98.5 x 10 reported by

C o r t in i and W ilk ie ( 1 9 7 8 ) . T h is f ig u r e in c lu d e s th r e e sm a ll

fra g m en ts w hich were r e s o lv e d by p o ly a c r y la m id e g e l

e le c t r o p h o r e s i s but n o t mapped.

- t i l -

The mean m .wt. o f th e S segm ent o f th e HSV-1 genome

i s 1 6 .8 ± 0 . 5 x 1 0 ° , and tha/t f o r HSV-2 i s 1 8 .6 x 1 0 °

(compare C o r t in i and W ilk ie , 1978: 1 7 .9 x 10 b f o r HSV-2).

On th e b a s i s o i t n e s e r e s u l t s i t seems t h a t th e two s e r o t y p e s

d i f f e r i n th e m .wt. o f S by 1 -2 x 10^ (1 5 0 0 -3 0 0 0 b p ) . I t i s

p o s s i b l e t h a t th e HSV-1 and HSV-2 L seg m en ts are o f eq u a l

s i z e , and t h e r e f o r e t h a t th e HSV-2 genome i s l a r g e r tn an t h a t

o f HSV-1 by 1 -2 x 10 i n m.wt* Morse e t a l . (1977) l i k e w i s e

m entioned t h a t HSV-2 DNA i s 3 x 1 0 ° l a r g e r i n m .w t. than

HSV-1 DNA, and R eyes e t a l . (19 7 9 ) i n d i c a t e d t h a t th e e x t r a

DNA i s i n S . The p r e c i s e l o c a t i o n o f th e e x t r a DNA i n th e

HSV-2 S segm ent ca n n o t be d eterm in ed from th e r e s u l t s

p r e s e n te d h e r e , but th e a l ig n m e n t o f r e s t r i c t i o n s i t e s

con c lu d ed from th e recom b in ant a n a l y s i s summarised i n F ig u re

C 2.13 s u g g e s t s t h a t th e major p r o p o r t io n i s l o e a t e u i n

HSV-2 BamHI 1 i n th e m idd le o f Ug. The f u n c t i o n o i tn e

a d d i t i o n a l DNA i s unknown.

The p h y s i c a l maps a l s o p r o v id e in fo r m a t io n r e g a r d in g

th e s i z e s o f in v e r t e d r e p e a t s , which i s summarised i n Table

C l . 1 3 . These r e s u l t s are i n agreem ent w ith p r e v io u s ly

p u b l is h e d e s t i m a t e s f o r HSV-1 and HSV-2. The s i z e o f

HSV-1 TRg ( i . e . c a ) has s in c e been more a c c u r a t e l y e s t im a te d

a s 6720 bp (m .w t. 4 .3 7 x 1 0 ° ) , from d e t a i l e d r e s t r i c t i o n

mapping and n u c l e o t i d e se q u e n c in g e x p e r im e n ts ( S e c t i o n 3

o f R e s u l t s ; M-J. M urchie, p e r s o n a l com m u n ica tio n ) .

Tables Cl.1-01.5

M o lecu la r w e ig h t s and r e l a t i v e m o l a r i t i e s o f

r e s t r i c t i o n fr a g m e n ts o f HSV-1 DNA produced by

Kpnl, BamHI, Khol and P v u I I , and o f HSV-2 DNA

produced by Bamli l .

A l l r e s t r i c t i o n fra g m en ts o f a d i g e s t co u ld n o t

be r e s o lv e d on a g e l o f s i n g l e c o n c e n t r a t io n , so

r e l a t i v e m o l a r i t i e s o f l a r g e r fra g m en ts were

e s t im a te d from lo w er c o n c e n t r a t io n g e l s , and

th o s e o f s m a l le r fra g m en ts from h ig h e r c o n c e n t r a t io n

g e l s . The r e s u l t s were l in k e d u s in g fra g m en ts o f

in t e r m e d ia t e s i z e w hich were r e s o lv e d on both

g e l c o n c e n t r a t i o n s .

Table Cl.l

HSV-1 t o I fr a g m e n t(s )

mol* w t. x 10“ 6

r e l a t i v e m o la r ity (± stan d ard d e v i a t i o n )

p r e d ic t e dr e l a t i v em o la r ity

abed 8 .0 2 .9 ( 0 .2 ) 3 .5e f g '7.0 2 .5 ( 0 . 2 ) 2 .5 'h 6 .0 1 .3 ( 0 .2 ) 1 .0i 5 .8 1 .0 ( 0 . 2 ) 1 .0d 5 .5 0 . 6 ( O . l ) 0 . 5k 4 .8 0 . 6 ( 0 . 1 ) 0 .51 4 .4 1 . 0 ( 0 . 1 ) 1 .0m 5 .2 1 .0 ( O . l ) 1 .0no 3 .0 2 . 0 ( 0 . 2 ) 2 .0p 2 . 8 1 .0 ( O . l ) 1 .0q 2 .4 1 .1 ( O . l ) 1 .0r 2 .4 1 .1 ( O . l ) 1 .0s 2 .2 1 .1 ( O . l ) 1 .0t 2 .0 1 .1 ( O . l ) 1 .0u 1 . 6 1 .3 ( 0 . 2 ) 1 .0V 1 .2 1 .2 ( O . l ) 1 .0wx 1 .1 2 . 4 ( 0 .4 ) 2 .0yz 1 .0 2 .3 ( 0 .3 ) 2 .0a 1 0 . 8 1 .0 ( 0 .2 ) 1 .0bl 0 . 5 l . l ( 0 .2 ) 1 .0c l 0 .4 2 1 .1 ( 0 . 2 ) 1 .0

Ta b le C l . 2

HSV-1 BamH I f r a g m e n t ( s )

m ol. wt, x 10“ 6

r e l a t i v e m o la r ity (± stan d ard d e v ia t io n )

p r e d ic t e dr e l a t i v em o la r i t y

abcdefghiJk1mnopars tuvwxyza'b!c ld!e 1£ ’g 1h i .i l<j~

1*mln l

8.06.26 .05 .65 .45 .0 4 .94 .8 4 .34 .23 .93 .53 .33 .22 . 42 . 22 . 0 1.81 .71 . 5 1 .3 1.1 0 .9 0 0 .6 3 0 . 5 8 0 .5 5 0 .5 1 0 .4 8 0 .4 5 0 .3 5 0 .1 4 0 .1 3 0.10 0 .0 6

1.01.01.01.01 .01 . 01 .01 . 01 . 01 . 01 . 01.01 . 01.01.02.01.02.02 . 01 . 04 .01.0 1.0 1.0 1 . 0 1.0 2 . 0 1.0 1.0 1.0 1.0 1.0 2 . 0 1.0

Table Cl. 3

HSV-1 Xho I f r a g m e n t ( s )

m ol. w t. x l c T 8

r e l a t i v em o la r ity(-s ta n d a r dd e v i a t i o n )

p r e d ic t e dr e l a t i v em o la r ity

a 1 0 .0 0 .7 ( 0 .1 ) 1 . 0b y.o 0 .9 ( 0 .1 ) 1 . 0c 6 .6 0 .7 ( 0 .0 ) 0 .5CL 6 .2 0 . 5 ( 0 .0 ) 0 .5e 5 .7 1 .3 ( 0 .0 ) 1 . 0f 5 .2 1 . 1 ( 0 .1 ) 1 . 0

g h i 4 .9 2 . 5 ( 0 . 2 ) 2 .5i 4 .6 0 . 6 ( 0 .0 ) 0 .5k 4 .0 1 .2 ( 0 . 1 ) 1 . 0run 2 .5 1 .8 ( 0 .2 ) 2 .00 2 .3 1 .2 ( 0 .1 ) 1 . 0p q r s t 2 . 1 4 .5 ( 0 .7 ) 5 .0u 1 .8 1 .0 ( 0 .1 ) 1 . 0

V 1 .5 1 .0 ( 0 .1 ) 1 . 0w 1 . 4 1 .1 ( 0 .1 ) 1 . 0

xyz 1 . 4 3 .0 ( 0 .1 ) 3 .0a 1 1 .4 0 .9 ( 0 .2 ) 1 . 0b* 1 .3 0 .9 ( 0 .1 ) 1 . 0c l 1 .2 0 .9 (0 .2 ) . 1 . 0

d* 1 .0 1 .1 ( 0 .3 ) 1 . 0e ! f 1 0 .8 3 2 .2 ( 0 .1 ) 2 .0:r 1e> - 0 .7 3 1 .1 ( 0 .2 ) 1 . 0hi i ' 0 .6 2 1 .7 ( 0 .1 ) 2 .0j ' 0 .5 4 1 . 0 ( 0 .1 ) 1 . 0k I 0 .4 3 0 . 8 ( 0 .0 ) 1 . 0l l m ’n 1 0 .3 7 2 .8 ( l .O ) 3 .0o ' p i

*i.

0 .3 0 1 .4 ( 0 .2 ) 2 .0

Table 01*4

riSV-1 Pvu I I f r a g m e n t ( s )

abcdef

h1jklmnoPq r s tuvwxyza 1b i c 1die*f ' .glh*i 1 .

j ' k ' l *m’n i o lp ’q l -

r * s 1t !u*V ’

w’x 1

m ol, wt, x 10“ b

17141295 .34 .23 .83 .73 .53 .22 .72 .2 2 . 01 .91 .71 .51 . 51 .41 .4 1 .3 1 .2 5 1 .11 .0 5 0 .9 0 .8 3 0 .8 0 0 .7 0 0 .6 0 0 .4 0 0 .3 8 0 .3 5 0 .3 3 0 .3 0 0 .2 8

r e l a t i v e m o la r ity (±stan d ard d e v i a t i o n )

p r e d ic t e d r e l a t i v e m o la r ity

0 .3 ( 0 .1 )0 .5 ( 0 .1 )0 .4 ( 0 .1 )0 .5 ( 0 .1 )1 .2 ( 0 .2 )0 .9 ( 0 .1 )1 .0 ( 0 .1 )0 , 6 ( o . i )1 .1 ( 0 .1 )3 .2 ( 0 .5 )1 .1 ( 0 .1 )1 .0 ( 0 .1 )1 .0 ( 0 .1 )4 .0 ( 0 .6 )1 .3 ( 0 . 2 )1 .2 ( 0 . 2 )1 .5 ( 0 .2 )0 .9 ( 0 . 1 )0 .9 ( 0 . 2 )0 .9 ( 0 .1 )0 .9 ( 0 . 1 )2 .1 ( 0 .2 )1 .8 ( 0 .1 )1 .0 ( 0 .1 )2 .1 ( 0 .1 )1 .0 ( 0 .0 )2 .9 ( 0 .4 )3 .3 ( 0 .2 )

0 . 50 .50 .50 . 51 .01 .01 .01 .01.04 .01.0 1.0 1.04 .01.0 1.0 1.0 1.0 1.0 1.0 1.0 2 . 0 2 . 0 1.0 2 . 0 1.03 .03 .02 .0 2 . 0 1.0 1.0 1.0 2 . 0

Table 01.5

HSV-2 JBamll I fr a g m e n t (s )

m ol. w t. x 10~5

r e l a t i v e m o la r ity ( t s ta n d a r d d e v i a t i o n )

p r e d ic t e dr e l a t i v em o la r i t y

abcdef6h i j k 1 m n op q.rs tuvwxyza 1b 1c 1 a 1

h'

J'k! l 1m* .

8 . 05 .24 .94 .74 .33 .73 .33 .12 .8 2 . 62 . 42 .32 .2 2 . 01 .9 1 .81 .71 .7 1 .61 .51 .3 1 .2 1 .1 51 .0 5 0 .9 6 0 .8 0 0 .7 0 0 .5 8 0 .4 7 0 .3 9

2 . 01 .01 .01 .01 .01 .03 .01 .0 1.0 1 .0 1.0 1.03 .01 .0 2 . 0 1 .0 2 . 0 1 .0 2.0 1 .0 1 .0 2 . 0 1 .0 1.0 2.0 1.0 1.0 1.0 2 . 0 2 . 0

Table Cl.6

D i g e s t i o n o f HSV-1 Kpn I fra g m en ts by o th e r

r e s t r i c t i o n e n d o n u c le a s e s . The summarised d a ta

a r e ta k en from F ig u r e s C l . 7 - 9 .

M o le c u la r w e ig h ts r e f e r to th e p ro d u ct fra g m en ts

o f r e c l e a v a g e o f p u r i f i e d Kpn I fr a g m e n ts .

L e t t e r s a r e g iv e n when a p ro d u ct fragm ent

c o r r e s p o n d s to a fragm en t produced by th e

r e s t r i c t i o n o f HSV-1 DhA w it h th e r e c l e a v i n g

e n d o n u c le a se a l o n e .

x t h i s fragm en t i s n o t r e c le a v e d

* t h i s fragm en t i s r e c l e a v e d , but t h e p ro d u ct

s i z e s a r e n o t known

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Table 01,7

D i g e s t i o n o f HSV-1 BamH I fra g m en ts T?y o t h e r

r e s t r i c t i o n e n d o n u c le a s e s . The summarised d a ta

a re from d o u b le d i g e s t i o n and r e c le a v a g e

e x p e r im e n ts . M o le c u la r w e ig h ts r e f e r to th e

p r o d u c t fr a g m e n ts o f r e c le a v a g e o f i s o l a t e d

BamH I f r a g m e n ts . L e t t e r s a r e g iv e n when a

p r o d u c t fra g m en t co rr e sp o n d s to a fragm en t

produced by th e r e s t r i c t i o n o f HSV-1 DNA w ith

the r e c l e a v i n g e n d o n u c le a se a lo n e .

x t h i s fragm en t was n ot c le a v e d

Table

CU

tf>a0■pHcd 000

•d • • •<d H l l ldo© 3( -3" CM SOS P| • • •

wl H CM X CM HG•H(0•Pa© »na •do o oa •u H VO H•

cdl o ^-p •o wl H Ol cm H X

•d0uQi

©«<d H •© HHO od •a Hi K <M K M H0■da«doa•H H> H Hflj H • •© H HH •dO at 00© •H • •a wl X X X 1 1

H

•d© Cut> cql<ti—©H T>O a© ©a & k 1 m n o

e C

l.7 (c

onti

nued

)

ndo4*rH(0•d<0toEd•Hm-pd®

<0U

+jodTJobp«

90}<09Oddo•dd®tod•H>(0<DHO•U

H

a

H

00o00o

a*.

HO

i lcm

1T\oVOooH

CM

OCM

Om-=r

(01 X X X

oooo

X X X

mCM

mcn

cn•o dPt m oH H

X H a

H

Ocn

x x

oin

X o x

a

m•if

inmX

&(0

H8

? 9> col <0"— ©H *2 dc*

Pi

■■i r'-ZS'iki

continued

over

leaf

Table

C1.7

(con

tinu

ed)

ndo■pH(4•da} H® G|B w H

wl X M X Xd•HQQ-Pa© in& c\t00 •d oH<H

<01 CM-P Pi •OH wl M H O H MPTJo(hn.

a)ma H0H «O °ld °\ Hd ml M M M Mo•dd<Ddod >-V•H H H► H «a) H o©H TfO -3“<D •HI M •U ml X X 0

j

X

-iK

•a 0|a) dl> mld^-'©rH d •*O d 110© du m e f h i

•HH

a

no>do•HV-POa<0•H

o|

I

Table Cl. 8

Summary o f HSV-1 Xho I d o u b le d i g e s t i o n d a t a .

Numbers i n p a r e n t h e s e s r e f e r t o th e number o f

m olar fr a g m e n ts w hich were c l e a v e d w i t h in a

m u lt i p l e band.

j/

Table Cl.8

secondendonuclease

Xho fragments which were cleaved in double digest

Xba I (a f h d')Hind III a e m ghi(0.5) pqrs(2) a* c*EcoR I a b c d f ghi(l.5) j mn(l)

pqrs(l) uBgl II a e f ghi(l) 1 mn(l) pqrs(3) uHpa I a b e f ghi(2) k 1 a» c* d*k e s I a b f k ran(l) o pqrs(3)

v w x y z e *BamH I a b o d e f g h i . j l m n

pqrs(3) v w xyz(2)

Table Cl.9

Summary o f HSV-1 Xho I fr a g m en ts to w hich i s o l a t e d

n i c k t r a n s l a t e d HSV-1 BamH I fra g m en ts h y b r id i s e d .

H y b r id i s a t io n o f BamH I k was n o t a s s p e c i f i c a s

f o r t h e o t h e r fr a g m e n ts , presum ably owing to

c o n ta m in a t io n by o t h e r fr a g m e n ts .

Table Cl,9

HSV-1 BamH I fragment

homologous fragments in HSV-1 Xho I digest

k (c d ghi j pqrs)1 bm 1n xyz c e io a xyzpq r c d f ghi j pqrs xyz e 1s o d pqrst f pqrsuv f pqrs j *w xyz ghi

Table Cl.10

Summary o f HSV-1 Pvu I I d ou b le d i g e s t i o n d a t a ,

lum bers i n p a r e n t h e s e s r e f e r to th e number o f

fr a g m e n ts w i t h in a m u l t ip l e band w hich were c l e a v e d .

Table Cl,10

secondendonuclease

Pvu XX bands which were cleaved in the double digest

Xba X c d f g j«k*l»(1)

Hind III c d g rst(l) x b*c’(l) d * e ' ( l )

EcoR X a b c d e f p q w y J ,k ,l ,(l)

Bgl II(bands larger than u)

a b e g h q rst(l)

Sea I a b c d e f g h l n b ,c ,(l)

Table Cl.11

D i g e s t i o n o f HSV—2 BamH I fra g m en ts by o t h e r

r e s t r i c t i o n e n d o n u c le a s e s . M o lecu la r w e ig h ts

r e f e r t o th e p r o d u c t fra g m en ts o f r e c le a v a g e

o f i s o l a t e d BamH I fr a g m e n ts , l e t t e r s a r e g iv e n

when a p r o d u c t fragm en t c o r r e sp o n d s to a fragm ent

produced by th e r e s t r i c t i o n o f HSV-2 DNA w ith

th e r e c l e a v i n g e n d o n u c le a se a l o n e .

x t h i s fragm en t was n o t r e c l e a v e d .

a)3o•H

H

3VO

n|o Bio »n •Hlcno

o Os• •H OS

9 H VO •ir\ O -=f O• «H O• CM-tf•P I H H cn H H H H

•H0a

•H

«P

•H9>Uat

o©

HO0■MO3*douPi

9<099

HO33o•d3©

fcjD3•rl>99Ho©U

300 c\* H

• • •H H H

CM CM H• • •

c n H c n CM

PIir\

<\*HlOcn

c n cn

-3-

HHH-d3•H«

Hr -

lT\CM

VO

CNJ

OloCM

HflS8 a

Ci

VTy

Cto •HA

Tab

le

C/JI

(co

nti

nu

ed)

ado

• r t

HH•rtBd•rtPf t•rt©>(44Hdo©HOa+>od*dodft

(D(04©HO§o•dd©

ood•H►cd©HO©

H

H

II

H

Os

rs

V\

-=f•

OOs CM

• « •O ITv o iH

»fN•

H OS• • »4 •

H H O ►4 r t a

00

ors

a

cm

o(VI

cm a h a a

a a> a n a a

SO

00 a

a* ft

p0) N

•d®>HOo®U

POd

x>

•d®

®

00d a 4 *e•rt

A-P 43|

-p 4 d ®jo •k- e

p p to n ® ® to to ■rt *H •d Tl© ©

•dd4

A *e•d p d d 4 ®a) | 6>

48

•d ® p 4 H Oa

CO

oo«

4>

*1Po

JOdo•d®Apd■rt4

43•d4}>4®

'do•d©Ap

pd©

&4h<H•d rt ©

O P © 4h H

Oa a 4 *H

441 Ap

•d

- d -431 43

4 •to

p pd •© ©I toto *rt 4 *d h<H ©

H© 43d d o o— 73

- , ©•dl Ap

« « 9 °« *d •*d 44 a

» S)

1 Vi dO •rt P

• ©to

H►>

4•rt*da 4 43© >* •p 4 •* u

0 © ©1 ©d rt >d

O© 9 *1© U CP

jdp d 4» 19ft

0ftd

4 4Ol

Table Cl>12

Summary o f HSV-2 BamHI fr a g m en ts to w hich i s o l a t e d

n ic k t r a n s l a t e d HSV-2 Hind I I I / BeoRI fr a g m e n ts

h y b r i d i s e d . The HSV-2 Hindi I I / EcoRI map i s from

W ilk ie e t a l . (1 9 7 9 b ) .

Table 01.12

HSV-2Hind/Bcofragment

homologous fragments in HSV-2 BamH I digest

1-6>

ab o e f g hij m o pst u vw yz a* e* h'

7-8 ab d hij n q9 ab k v10-11 c f g p vw y z

12 d hij i»13 g p u vw y z a * e 114-15 c g p u vw x yz a*16 1» c *d*17 1 e * f«18 f k19 st20 r21 b*22 ab23 f

0 0.2 0.4 0.6 0.8 1.1 1 1 1 1, li

l l l II i II i it i it • i i i i10 15 8 12 2019 7 22 6 5 24 9 18 23 11 13 16 21 14

Table Cl.15

Estimates of upper and lower limits for molecular weights of the repeat sequences of HSV-1 and HSV-2.Molecular weights are given in millions and the relevant fragments are quoted.

ftH+ r On

CO • CM • Hft o • o •H iH H C>*

•P S \ y,-^ Qfl5 0) ol 51 04 Mft © 0 (0 <d 5© o jq ft ft ©u c >< B B «© V Voo 3ti a* H c\i ONo © • • • •H (0 vo vo ca

— sr tl

91 S I+> *A fcdcd 51 51<1) - 4 * 1a 9 s© © o 0 4 (0fn O fl o a PQ

a W H w V-/4J © v_Ph 3 IT\ XAo a* VO IT\ vo rHB © • • • •(0 © ca CA

u u u© © © ©A > ft0< o ft O3 H 3 H

H e*1 1f>co COB B

llgure 01>1

M o le c u la r w e ig h t c a l i b r a t i o n o f HSV-1 DNA r e s t r i c t i o n

fr a g m e n ts (1 -1 0 x 10^ d a l t o n s ) .

1 . Lambda DNA c le a v e d w ith Hind I I I .

2 . Lambda DHA c le a v e d w ith KcoK I .

3 . HSV-1 DMA c le a v e d w ith Xho I .

4 . HSVr-1 DNA c l e a v e d w ith BamH I .

5 . HSV-1 DNA c l e a v e d w ith Kpn I .

6 . HSV-1 DNA c le a v e d w ith Hpa I .

Ihe 0.5>» a g a r o se g e l c o n t a in in g e th id iu m bromide

was photographed under UV i l l u m i n a t i o n . M o lecu la r ]

w e ig h t s (x 10"6 ) o f lambda DNA fr a g m e n ts a r e i n d i c a t e d j

(Thomas and D a v is , 1975; Murray and Murray, 1 9 7 5 ) .

1 2 3 4 5 6

13.7 -

6 .4 -

4 .7 -4 .3 -3.7 _ 3.5 “3 .0 -2.9 -

2.1 -

1.6 -1.4 -

Pigure Cl.2

M o le c u la r w e ig h t c a l i b r a t i o n o f HSV-1 and HSV-2 DNA

r e s t r i c t i o n f r a g m e n ts (Q .1 - 1 * 0 x 10 d a l t o n s ) .

M. J& 174 DNA c l e a v e d w i t h H i n d l l .

1 . HSV-tl DNA c l e a v e d w i t h BamHI .

2 . HSV-1 DNA c l e a v e d w i t h X h o l .

3 . HSV-2 DNA c l e a v e d w i t h BamHI.

)&C174 DNA was ^ P - l a b e l l e d by n i c k t r a n s l a t i o n and

HSV-1 and HSV-2 DMA w ere 32P - l a b e l l e d i n v i v o .

E l e c t r o p h o r e s i s was c a r r i e d o u t on a 1 . 7 # a g a r o s e

g e l w h ich was d r ie d t h e n a u t o r a d io g r a p h e d .

M o le c u la r w e i g h t s (x 10 o f 0X174- DNA fr a g m e n t s

a r e i n d i c a t e d .

0.681-

0.501-

0.396-0.322-0.255-0 . 220 -0.190-

0.137-i

0.681

0.501

0.3960.3220.255

- 0.220-0.190

0.137

Figure Cl. 5

K e s t r ic t io n p r o f i l e s o f HSV-1 (u p p er) and HSV-2

DNA (lo w er ) w hich were P - la b e l l e d i n v iv o .

E le c tr o p h o r e s is was c a r r ie d ou t on 0 .5 # (upper4 ^

p o r t io n s ) and 1 .2 # a g a r o se ( lo w er p o r t io n s ) .4 ^

Bands a re in d ic a te d o n ly f o r th o se d i g e s t s f o r w hich

r e s t r i c t i o n maps are a lr e a d y known, o r were

deduced i n t h i s work.

EcoR

1

Bgl

II

Hpa

1

Kpn

1 ZIm Xh

o 1

cf• I 9

II IB = ab

cde — a— b9hi ® J j — f a d — a

Jk1

— k1 ----i 'j = § S

■ h P . III!

m— i f— m «• jk1- k Jk — n — m Jk

1 o nm *mn P — no — n i

m P9r■ ■ or s[

s topq

_mno- P t s

o

— n

u

- - V— wx

st

— uv— w

u__ Vw • xyz a'

b— c

o U — VZ V a

ab d

— e' f

cdghi'

bhf9i

ic' k

j I'm'q

n'op

%cd

f9fh

oSsw*Vab'c'de

-&•Amno'pqr . s t

I I

Figure Cl«4

R e s u lt s o f r e c le a v a g e o f i s o l a t e d HSV-1 X b a l,

H in d III and EcoRI r e s t r i c t i o n fragm en ts by KpnI .

N ick t r a n s la t e d fragm en ts were c le a v e d and th e

p r o d u cts e l e c t r o p h o r e t ic a l ly se p a r a ted on 0 . 5 %

(upper p o r t io n s ) and 1 *5# a g a r o se g e l s ( lo w er

p o r t io n s ) , w hich were th en d r ied and a u to r a d io

graphed . The i d e n t i t y o f the p a ren t fragm ent

i s g iv e n a t th e to p o f each tr a c k , and p o s i t i o n s

o f HSV-1 KpnI fragm en ts are in d ic a te d in th e

d im en sion o f e le c t r o p h o r e s i s . C leavage p r o d u cts

are marked to th e r ig h t o f each t r a c k .

m

i i

£9

CCcuUJ

5 -x (0

i

f t

i9

I II 1 I I35—— Jt —I t

III II I 1£ 0 a sv*..

si i i M i i i l l i i i i£g a -M-

25 a?(5

— c ° P Nre a; x — Ji — E c a. crj *- 3 > > >> J. >

0)■3

<0X)X

' i i ' "i 0 i 6 ' 1A >0 0 0 0 00 0 0 0

0 0 0 o 0 0 0

ooo 0

Figure Cl,5

R e s u l t s o f r e c le a v a g e o f i s o l a t e d HSV-1 B g l l l

and Hpal r e s t r i c t i o n fr a g m e n ts by KpnI ,

D e t a i l s a r e g iv e n i n th e le g e n d t o F ig u r e C l . 4 .

Figure Cl.6

Combined r e c le a v a g e d a ta o f t h e HSV-1 genome f o r

Kpn I . The s c a l e i s o f m o le c u la r w e ig h t (x 1 0 “ ^ ) .

HSV-1 Kpn I fr a g m e n ts p rod u ced by r e c le a v a g e w ith

Kpn I a r e l i s t e d u n d er ea ch p a r e n t fr a g m e n t , a s

w e l l a s th e m o le c u la r w e ig h t s (x 10~^ ) o f r e c le a v a g e

fr a g m e n ts n o t c o r r e s p o n d in g to HSV-1 Kpn I fr a g m e n ts .

The sym b ol □ i n d i c a t e s fr a g m e n ts n o t c le a v e d by

Kpn I . R e c le a v a g e p r o d u c ts o f j o i n t fr a g m e n ts a re

shown on th e r i g h t . The Kpn I map d ed u ced from

t h e s e d a ta i s shown a t th e b o tto m .

cn ' r~> ,

1—i-rrt f

^

Figure 01.7

D ou b le d i g e s t s o f i n v iv o -*2P - l a b e l l e d HSV-1 DNA.

1 . Xba I .

2 . Xba i/K p n I .

3 . Kpn I .

4 . Hind I I I /K p n I

5 . Hind I I I . '

6 , EcoR I .

'7. EcoR I/K pn I .

8 . Kpn I .

9 . i t e l II/K p n I .

1 0 . B g l I I .

1 1 . Hpa I .

1 2 . Hpa 1 / Kpn I .

1 3 . Kpn I .

E le c t r o p h o r e s i s was c a r r ie d o u t on a 0 . 6 # a g a r o s e4

g e l w h ich was th e n d r ie d and a u to r a d io g r a p h e d .

HSV-1 Kpn I r e s t r i c t i o n fr a g m e n ts a r e i n d i c a t e d .

1 2 3 4 5 6 7 8 9 10111213

- • I s ! 1 ! ! ? * - ; f p - a b c d

m Z \

i n i i s r

mm K - m

r r S i r0

z ^ r = s t! i

•' — -U

m * »

-v-wx-yz

-a

b

c1

Figure Cl ♦ 8

C le a v a g e o f HSV-1 Kpn I r e s t r i c t i o n fr a g m e n ts

w ith v a r io u s r e s t r i c t i o n e n d o n u c le a s e s .

H ick t r a n s la t e d fr a g m e n ts w ere c le a v e d and th e

p r o d u c ts e l e c t r o p h o r e t i c a l l y s e p a r a te d on 1 #

a g a r o s e g e l s , w h ich w ere th e n d r ie d and a u to r a d io

g r a p h ed . The Kpn I fr a g m e n ts c le a v e d a r e

in d ic a t e d a t th e t o p * o f th e t r a c k s , w ith th e

r e c l e a v i n g e n d o n u c le a se a b o v e ea ch p a n e l . The

m arker d i g e s t o f th e r e c l e a v in g e n d o n u c le a se i s

in d ic a t e d i n th e d im e n s io n o f e l e c t r o p h o r e s i s .

H ot a l l r e c le a v a g e s a xe show n, b u t a l l th e d a ta

h a s b een in c lu d e d i h T ab le C l . 6 .

ccooLU

do uLU

I>1ri

M6 -8p-B

m

l i tB 1 1

■

CO-QX

do uLU

P-B ■ ■ aIi

To>

Figure Cl,.9

C le a v a g e o f HSV->1 Kpn I r e s t r i c t i o n fr a g m e n ts w ith

v a r io u s r e s t r i c t i o n e n d o n u c le a s e s . D e t a i l s a r e

g iv e n i n th e L egend to F ig u r e C l . 8 .

Bgl II Hpa I

CU <D _C

I !opqr

.

" •

EcoR I Hpa I

a-n

opqr

Figure 01,10

C ro ss-" b lo t o f HSV-1 Hpal a g a in s t n ic k t r a n s la t e d

HSV-1 KpnI . The u n la b e l l e d d im e n s io n i s

in d ic a t e d by th e m arker s t r i p t o th e r i g h t , and

th e l a b e l l e d d im e n s io n a c r o s s th e t o p . F a in t

s p o t s n o t c o r r e s p o n d in g t o KpnI bands a r e due

t o some fr a g m e n ts r e s u l t i n g from s l i g h t

u n d e r d ig e s t io n o f th e DHA.

— O f- u.I cc-Q O s - O) — — — fc C o

i w n 1 1 1 * i i •

> N -

5* -> -

Ca

Figure Cl.11

Summary o f HSV-1 KpnI h y b r i d i s a t i o n d a t a , u s in g* - -

th e p h y s i c a l map d ed u ced from r e c le a v a g e

e x p e r im e n ts . B e n e a th e a c h fra g m en t i s w r i t t e n

th e fr a g m e n t o f th e o t h e r d i g e s t w ith w h ich i t

h a s h om ology , a s d ed u ced from F ig u r e C l . 1 0 .—6The s c a l e i s m o le c u la r w e ig h t (x 10 ) .

iRZHfffrvuRgmnnn; innru-drawni t i i i i m f i n

Figure 01>12

D e te r m in a t io n o f th e r e l a t i v e o r d e r o f HSV-1

Kpn I n and p . The r e s t r i c t i o n d i g e s t p r e s e n t on

th e n i t r o c e l l u l o s e s t r i p s i s n o te d a t th e to p , and

th e fr a g m e n ts o f th e d i g e s t t o th e l e f t o f e a ch

p a n e l . Bands to w h ich h y b r id i s a t io n o f th e

n ic k t r a n s la t e d DHA p rob e o c c u r r e d a r e shown to

th e r i g h t o f ea ch p a n e l .

The p r o b e s w ere a s f o l l o w s . .

1 . HSV-1 ( l e f t p a n e l) o r HSV-2.DNA ( r i g h t p a n e l ) .

2 . HSV-1 Kpn I n o .

3 . HSV-1 Kpn I p .

HSV-1 Hpa I

ab

cdef f c

9 h _> 3j m •<- ■I

m n

opqr

• b

(i)

t • u ■

HSV-2 Bgl II

q|

r i

L *.

2 3

1 2 3

Figure Ql.13

Combined r e c le a v a g e d a ta o f th e HSV-1 genome f o r

BamH I . The s c a l e i s o f m o le c u la r w e ig h t (x 1 0 " ^ ) .

HSV-1 BamH I fr a g m e n ts p rod u ced by r e c le a v a g e w ith

BamH I a r e l i s t e d u n d er ea ch p a r e n t fr a g m e n t , a s

w e l l a s th e m o le c u la r w e ig h ts (x 1 0 ““^) o f r e c le a v a g e

fr a g m e n ts n o t c o r r e s p o n d in g t o HSV-1 BamH I fr a g m e n ts .

The sym bol □ i n d i c a t e s fr a g m e n ts n o t c le a v e d by

BamH I . R e c le a v a g e p r o d u c ts o f j o i n t fr a g m e n ts

a r e shown on th e r i g h t . The BamH X map ded u ced

from t h e s e d a ta i s shown a t th e b o tto m .

LO LO *“iOmm r \io co <n *— o

04 on CO 04

Figure Cl.14

Summary o f HSV-1 BamH I h y b r id i s a t io n d a ta , u s in g

th e p h y s i c a l map d educed from r e c le a v a g e

e x p e r im e n ts . B en eath e a c h fra g m en t i s w r i t t e n th e

fra g m en t o f th e o th e r d i g e s t w ith w h ich i t h a s

h o m ology . . For th e BamH I map, th o s e fr a g m e n ts

u n d e r lin e d r e f e r to hom ology w ith Hpa X fr a g m e n ts ,

and th e o t h e r s r e f e r to th e Kpn I map.

.

—

Figure Cl.15

Combined r e c le a v a g e d a ta o f th e HSV-1 genome £ o r

Xho I . The s c a l e i s o f m o le c u la r w e ig h t (x 1 0 ““^*).

HSV-1 Xho I fr a g m e n ts p rod u ced by r e c le a v a g e w ith

Xho I a r e l i s t e d u n d er e a c h p a r e n t fr a g m e n t , a s

w e l l a s th e m o le c u la r w e ig h ts (x 10~^ ) o f r e c le a v a g e

fr a g m e n ts n o t c o r r e s p o n d in g to HSV-1 Xho I f r a g m e n ts .

The sym bol 0 i n d i c a t e s fr a g m e n ts n o t c le a v e d by

Xho I . H e c le a v a g e p r o d u c ts o f j o i n t fr a g m e n ts

a r e shown on th e r i g h t . The Xho I map d ed u ced


ip- «> h

CMt O

-..CpCM^oo

cnc m *"—■

oo in> p o

Figure 01.16


Pvu I I . The s c a l e i s o f m o le c u la r w e ig h t (x 1 0 ~ b) •

HSV-1 Pvu I I fr a g m e n ts p rod u ced by r e c le a v a g e w ith

Pvu I I a r e l i s t e d u n d er ea c h p a r e n t fr a g m e n t , a s

w e l l a s th e m o le c u la r w e ig h t s (x 1 0 ““^) o f r e c le a v a g ea

fr a g m e n ts n o t c o r r e s p o n d in g to HSV-1 Pvu I I fr a g m e n ts .

The sym bol C3 i n d i c a t e s fr a g m e n ts n o t c le a v e d by

Pvu I I . K e c le a v a g e p r o d u c ts o f j o i n t fr a g m e n ts

a r e shown on th e r i g h t . The Pvu I I map d educed

from t h e s e d a ta i s sh o rn a t th e b o tto m .

>N N

to pp. CNI

Figure 01.17


BamH I . The s c a l e i s o f m o le c u la r w e ig h t (x 10” ^ )•

HSV-2 BamH I fr a g m e n ts p rod u ced by r e c le a v a g e w ith

HatnH I a r e l i s t e d u n d er e a c h p a r e n t fr a g m e n t , a s

w e l l a s th e m o le c u la r w e ig h t s (x 1 0 “ ^) o f r e c le a v a g e

fr a g m e n ts n o t c o r r e s p o n d in g to HSV-2 BamH I fr a g m e n ts .

The sym bol O i n d i c a t e s fr a g m e n ts n o t c le a v e d by

BamH I . K e c le a v a g e p r o d u c ts o f j o i n t fr a g m e n ts

a r e shown on th e r i g h t . The BamH I map d ed u ced


C A J MVf'AN): ?:■] r-"T

HSV-2

a i L i £ |:- T-ri; -.: ~;— j b ______ 1. ; . . . . . J , , . b

! — =i-— ,L.....

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tc?

jiIv ; :V. "q . s( :

m1 ■; -

J __

B a t II

: m i

d r p g

...... . , .„'... . □ i s

r ii o

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L _ .^ L □ S1 -6 0 - 4 0 - 2

0-1

9Hind HI/ B g t l l o 0-62 - 2 0 4

7

m1 - 5 1>2

1- 1-6i 1 i i in

l i i ‘) r s l n n T b

tkuUUJ

Figure Cl .18

A lig n e d r e s t r i c t i o n maps f o r HSV-1 BNA.

The s c a l e i s f r a c t i o n a l genom e l e n g t h .

The maps f o r Kpn I , BamH I , Xho I and Pvu I I

w ere d e te r m in e d a s a r e s u l t o f t h i s s tu d y ,

and t h e o t h e r s w ere th e work o f W ilk ie ( 1 9 7 6 ) .

Figure 01.19

A lig n e d r e s t r i c t i o n maps f o r HSV-2 DNA.

The s c a l e i s f r a c t i o n a l genome l e n g t h .

The map f o r BamH I was d e te r m in e d a s a r e s u l t

o f t h i s s t u d y , and th e o t h e r s w ere th e work

o f C o r t in i and W ilk ie ( 1 9 7 8 ) .

L ___

— ----------------

78

57

82

43

R e s t r i c t i o n maps f o r HSV-1 BNA a l ig n e d w it h

t h o s e f o r HSV-2 BNA. B a ta u se d t o d e te r m in e th e

b e s t a l ig n m e n t in c lu d e d r e s t r i c t i o n fr a g m e n t

s i z e s , i n t e r t y p i c h y b r i d i s a t i o n ( S e c t io n 2 o f

R e s u l t s ) , and a n a l y s i s o f i n t e r t y p i c r e c o m b in a n ts

( P r e s t o n e t a l . , 19785 Stow and W ilk ie , 1 9 7 8 ;

M arsden e t a l . , 1 9 78;. C h artran d e t a l . , 1979

and 1 9 8 0 ; S e c t io n 2 o f R e s u l t s ) .£

S in c e th e HSV-2 genpme h a3 an e x t r a 1 - 2 x 10

d a l t o n s o f BNA i n th e S seg m en t com pared w it h

t h a t o f HSV-1, th e f o l l o w i n g fr a g m e n ts h ave

b een s h o r te n e d i n th e HSV-2 m aps: Xba I J,,

Hind. I l l 1 , Ecoii I a , B g l I I 1 , Haa I ( g j ,

Kpn I &, and BamH I 1 . The s c a l e show s

f r a c t i o n a l genom e l e n g t h , and th e n a tu r e o f

j o i n t fr a g m e n ts i s shown on th e r i g h t .

S3CTI0H 2

HOBO 10 GY BiSWiSJSN HiSETBSVIHUS G3N0M3S

- 82 -

RESULTS


HSV-1 and HSV-2 sh are hom ology in ab ou t 5 0 £ o f t h e i r

DM se q u e n c e s ( K ie f f aJ a i « , 1 9 7 2 ) , w h ereas th e DM o f

p se u d o r a b ie s v ir u s (PKV) h as been r e p o r te d to be l e s s than

8 # hom ologous to HSV-1 and HSV-2 DM (B ronson e t a l . , 1 9 7 2 ) .

The p u rp o se o f th e work d e s c r ib e d h e r e was f i r s t l y to

d eterm in e th e genom ic d i s t r ib u t io n o f hom ologous seq u e n c e s

betw een HSV-1 and HSV-2, and to a s c e r t a in w h eth er th e genom es

a re c o l i n e a r . Second , e x p er im en ts were c a r r ie d ou t to

d eterm in e w h eth er r e g io n s o f g r e a te r hom ology betw een HSV-1

and HSV-2 DM a re a ls o hom ologous to PRV DNA and eq u in e h er p e s

v ir u s ty p e 1 (EHV-1) DNA to a g r e a te r e x te n t than o th e r r e g io n s .

Homology betw een th e HSV and human c y to m e g a lo v ir u s (CMV)

genom es was a ls o in v e s t i g a t e d . T h ird , c o l i n e a r i t y o f th e

genom es o f IISV and EHV-1 or PRV was c o n s id e r e d . L a s t , a

s tu d y was made o f a la r g e number o f HSV-l/HSV-2 in t e r t y p i c

reco m b in a n ts in o rd er to d eterm in e th e r e la t io n s h ip betw een

c r o s s o v e r lo c a t io n and r e g io n s o f h ig h e r hom ology .

D is t r ib u t io n o f hom ologous seq u en ces betw een HSV-1 and HSV-2 DNA

B lo t h y b r id is a t io n .p r o v id e s a good method f o r sem i-

q u a n t i t a t iv e e s t im a t io n o f th e p r o p o r t io n o f probe w hich

h y b r id is e s to each r e s t r i c t i o n fragm en t o f a genom e.

H y b r id is a t io n in s o lu t io n i s q u a n t i t a t iv e , but r e q u ir e s each

fragm en t to be i s o l a t e d and h y b r id is e d s e p a r a t e ly , and

th e r e fo r e i s much l e s s c o n v e n ie n t f o r i n i t i a l d e te r m in a t io n

o f th e d i s t r i b u t i o n o f hom ology betw een r e g io n s o f HSV-1 and

nSV-2 DNA.

F ig u r e s C 2 .1 -C 2 .2 show exam ples o f th e r e s u l t s o f

h y b r id is in g HSV-1 or iISV-2 p ro b es to n i t r o c e l l u l o s e f i l t e r s

c o n ta in in g fra g m en ts o f HSV-1 or HSV-2 DNA. The p rob es in

- 83 -

t h e s e , and su b se q u e n t, ex p er im en ts were p rep ared by n ic k Q

t r a n s la t io n i n th e p r e se n c e o f 10 g /m l DNase, and under

th e s e c o n d it io n s th e mean s i z e o f s in g le - s t r a n d e d DNA

fra g m en ts i s 400 n (R igby e t a l . , 1 9 7 7 ) . Each b lo t s t r i p

o f a p a ir c o n ta in e d an eq u a l amount o f DNA ( 0 .5 - 1 .0 yug), and

r e s u lt e d from t r a n s f e r o f DNA from th e same g e l . An eq u a l

amount o f probe DNA ( 0 .1 - 0 .2 yug) was added to each s t r i p -» ^

and a llo w e d to a n n e a l to s a tu r a t io n (5 -5 d a y s) under s t r in g e n t

h y b r id is a t io n c o n d it io n s (3 x SSC and D en h a rd t’ s s o lu t io n in

50# form am ide a t 45 ) • The e x p e r im e n ta l d e s ig n en sured th a t «

p o t e n t i a l c a u s e s o f a r t e f a c t s , su ch a s i n e f f i c i e n t t r a n s f e r

o f some r e s t r i c t i o n fra g m en ts in th e b l o t t i n g p r o ced u re ,

were c a n c e l le d o u t .

The e x t e n t o f in t e r t y p i c hom ology can in p r in c ip le be

d eterm in ed f o r each r e s t r i c t i o n fragm en t by com parison o f

th e band i n t e n s i t i e s produced by hom ologous and h e te r o lo g o u s

p r o b e s . In F ig u re C 2 .1 , f o r exam ple, HSV-1 BamHI o and r

are more in t e n s e than piq w ith HSV-2 p r o b e . A se m i-

q u a n t i t a t iv e e s t im a te o f h y b r id is a t io n to each fragm en t was

made by a u to d e n s ito m e tr ie sca n n in g o f s e v e r a l a u to r a d io g r a p h s .

Peaks were c u t o u t and w eigh ed , and th e f o l lo w in g r a t i o s

c a lc u la t e d .

R1 - wt o f peak w ith h e te r o lo g o u s probe wt o f peak w ith hom ologous probe

^2 — t o t a l wt o f p eak s w ith h e te r o lo g o u s probe t o t a l wt o f peaks w ith hom ologous probe

K3 = % /H g .

R'5 i s in d e p en d e n t o f s p e c i f i c a c t i v i t i e s o f th e p rob es and

d u r a tio n o f a u to r a d io g r a p h ic ex p o su re in th e l in e a r range o f

th e f i l m , and i s an a r b it r a r y v a lu e f o r r e l a t i v e hom ology.

- 84 -

A ll th e fra g m en ts o f r e s t r i c t i o n d i g e s t s c o u ld n o t be r e s o lv e d

on a s in g l e g e l , so i t was n e c e s s a r y to scan two a u to r a d io g r a p h s!

one from a g e l o f lo w er a g a ro se c o n c e n tr a t io n f o r bands o f

la r g e r m o le c u la r w e ig h ts , and one o f h ig h e r c o n c e n tr a t io n f o r

bands o f s m a lle r m o le c u la r w e ig h t . T h is approach le d to two

i n t e r n a l l y c o n s i s t e n t s e t s o f R^ v a lu e s i n each c a s e . A few

bands o f in te r m e d ia te m o le c u la r w e ig h t were r e s o lv e d on b oth

g e l s , so R v a lu e s f o r s m a lle r fr a g m en ts w ere m u lt ip l ie d by

a f a c t o r w hich e q u a lis e d th e two s e t s o f R^ v a lu e s f o r th e

in te r m e d ia te bands, th e re b y l in k in g th e two s e t s o f d a ta .

R- v a lu e s a r e , o f c o u r s e , m erely an a p p rox im ate i n d ic a t io n o f

r e l a t i v e hom ology, th e in t e r t y p ic genom e-to-genom e hom ology

b e in g u n i t y .

F ig u r e C 2 .3 shows h i s t o g r a p h ic a l ly th e r e s u l t s o f

s e v e r a l e x p e r im e n ts , and sum m arises th e r e g io n s o f g r e a t e r

hom ology betw een HSV-1 and HSV-2 DNA. There i s one r e g io n

o f g r e a t e r hom ology in TR g/lR g, and s i x , o r p erh ap s s e v e n ,

i n U^. R eg io n s o f l e a s t hom ology in c lu d e f e j / l R ^ , th e

ju n c t io n s betw een Ug and TRg/IRg, and th e c e h t r a l p o r t io n o f Ug.

F ig u re C2.2 shows th e e f f e c t o f tem p era tu re o f

h y b r id is a t io n upon h y b r id is a t io n o f HSV-2 probe to HSV-1

r e s t r i c t i o n fr a g m e n ts . The p a t t e r n s a t t h r e e d i f f e r e n t

tem p era tu res a re s im i la r , e x c e p t t h a t HSV-2 DNA h y b r id is e d

more e f f i c i e n t l y to th e j o in t and S te r m in a l fra g m en ts o f

HSV-1 DNA (BamHI k and g,) a t th e h ig h e r te m p e r a tu r e s . The

g e n e r a l s a tu r a t io n l e v e l o f HSV-2 probe to HSV-1 DNA

d e c r e a se d w ith in c r e a s in g te m p e r a tu r e .

C o l in e a r i t y o f th e genomes o f HSV-1 and HSV-2

I t h as been assumed in in t e r p r e t in g th e r e s u l t s o f

hom ology e x p er im en ts th a t th e genom es o f HSV-1 and HSV-2 a re

c o l in e a r . To t e s t t h i s assu m p tion p la sm id s c o n ta in in g HSV-2

- 85 -

H in d l l l fra g m en ts were n ic k t r a n s la t e d and h y b r id is e d to b lo t

s t r i p s c o n ta in in g BamHI fra g m en ts o f HSV-1 or HSV-2 DNA

(F ig u r e C 2 .4 ) . Fragm ents com plem entary to ea ch probe a re

l i s t e d in T ab le C 2 .1 . No h y b r id is a t io n o f p ro b es to r e g io n s

o f th e genome in w hich th e y do n o t map was d e t e c te d in t h i s

e x p e r im e n t. The few f a i n t bands o b serv ed when HSV-1 BamHI

b lo t s t r i p s were u sed (F ig u r e C 2 .4 ) were p ro b a b ly due to

s l i g h t u n d e r - d ig e s t io n o f th e HSV-1 DNA.

These r e s u l t s a re c o n s i s t e n t w ith c o l i n e a r i t y o f th e

HSV-1 and HSV-2 genom es. There i s no e v id e n c e from t h e s e

ex p e r im e n ts f o r hom ology betw een one r e g io n o f a genome and

a r e g io n o f th e same o r th e h e te r o lo g o u s genome m apping a t

a d i f f e r e n t p o s i t i o n .

N o n -c o lin e a r hom ology betw een th e genom es o f HSV-1 and HSV-2

The r e s u l t s o f c r o s s - b l o t e x p er im e n ts s u g g e s te d t h a t

c e r t a in fra g m en ts o f one genome a l s o h y b r id is e d to d i s t a l

fra g m en ts o f th e same, or th e h e t e r o lo g o u s , genom e. In

F ig u re C l .10 th e r e i s e v id e n c e o f hom ology betw een HSV-1

Kpnl i and k ( th e S te r m in i) and HSV-1 Huai m ( th e L term in u s)

and o p q r . In F ig u re C 2 .5 , HSV-1 Kpnl ^ and k h y b r id is e d to

HSV-1 BamHI d , j and s , and p erh ap s to b , c and e . Homology

betw een L and S te r m in i i s e x p la in e d by th e p r e se n c e to th e

a seq u en ce a t both te r m in i and a t th e L-S j o i n t . H y b r id is a t io n

to HSV-1 BamHI ^ was th e r e s u l t o f BamHI E (BamHI k w ith

an a d d i t io n a l a seq u en ce ) w hich co m ig ra ted w ith t h i s fra g m en t.

F ig u re C 2.5 shows a c r o s s - b l o t o f HSV-1 BamHI a g a in s t HSV-1

BamHI, and d em o n stra tes some h y b r id is a t io n o f HSV-1 BamHI

k and pq, to d . F ig u re C 2.6 shows some h y b r id is a t io n o f

KcoHI k to 1 . These r e s u l t s s u g g e s t a r e g io n in HSV-1 BamHI

d and BcoRI 1 to w hich th e S term in u s h as some seq u en ce

hom ology. H y b r id isa t io n betw een th e S term in u s and th e

- 8 6 -

ju n c t io n s betw een U- and TR^/IR^ was d e te c te d in some, but

n o t a l l , e x p e r im e n ts .

F ig u re C 2 .6 shows a c r o s s - b l o t betw een HSV-1 BamHI

and HSV-2 BamHI . The m ost o b v io u s a b e r r a n t h o m o lo g ie s a re

th o s e betw een HSV-1 BamHI k and pq and HSV-2 BamHI p and y z .

F a in te r s p o t s in c lu d e HSV-1 BamHI k and pq w ith HSV-2 BamHI

c , f , 1 and a 1, and HSV-2 BamHI a 1 w ith HSV-1 BamHI b, d and e .

Many, o f t h e s e a b er r a n t h o m o lo g ie s in v o lv e r e p e t i t i v e seq u e n c es

o r t h e i r ju n c t io n s w ith u n iq u e r e g io n s .

Homology betw een th e HSV genome and th e genom es o f EHV-1 and PRV

F ig u re C 2.7 shows th e r e s u l t s o f h y b r id is in g n ic k

t r a n s la t e d HSV-1, HSV-2, EHV-1 o r PRV DNA to b lo t s t r i p s

c o n t a in in g r e s t r i c t i o n fra g m en ts o f HSV-1 o r HSV-2 DNA.

T a b les C 2 .2 -C 2 .3 sum m arise th e HSV fra g m en ts to w hich EHV-1

and PRV probe h y b r id is e d . The r e g io n s o f hom ology o f e i t h e r

EHV-1 o r PRV DNA w ith th e genom es o f HSV-1 and HSV-2 are

shown in th e upper p a n e l o f F ig u r e C 2 .1 0 , and a r e l a r g e ly

s im i la r in p o s i t i o n to th e more hom ologous r e g io n s betw een

HSV-1 and HSV-2 DNA.

In o rd er to d eterm in e th e r e l a t i v e mapping p o s i t i o n s o f

HSV DNA se q u e n c e s hom ologous w ith EHV-1 and PRV DNA, n ic k. i

t r a n s la t e d p la sm id s c o n ta in in g HSV DNA fra g m en ts were

h y b r id is e d a t 40° to b lo t s t r i p s c o n t a in in g EHV-1 ..or PRV

r e s t r i c t i o n d i g e s t s . The r e s u l t s a r e shown in F ig u r e s C 2 .8 -

C 2.9 , and a r e summarised i n T a b le s C 2 .4 -C 2 .5 . The m id d le

and lo w e r p a n e ls o f F ig u re C 2.10 show th e p o s i t i o n s in th e

genom es o f EHV-1 and PRV to w hich ea ch c lo n e d fragm ent

h y b r id is e d . In in s t a n c e s where h y b r id is a t io n o ccu rred to a

band c o n ta in in g more than one r e s t r i c t i o n fra g m en t, r e s u l t s

o f th e o th e r d i g e s t s were u sed to d e term in e to w hich o f th e

fra g m en ts h y b r id is a t io n had o c c u r r e d . For exam ple, HSV-2

- 87 -HindI I I h h y b r id is e d to PRV Kpnl j k , but to BamHI k, in d ic a t in g

th a t h y b r id is a t io n was to Kpn l k r a th e r than to £ . The f a i n t

bands p r e s e n t on th e PRV DNA b lo t s t r i p s (P ig u r e C 2.9

such a s th o se j u s t below BamHI i . i and Kpnl 1 , w hich have n o t

been in c lu d e d in T ab le C 2 .5 , were due to th e p r e se n c e o f

m inute am ounts o f c o n ta m in a tin g pAT153 DNA in th e d y e - P ic o l l

w hich was added to th e d ig e s t e d PRV DNA p r io r to e le c t r o p h o r e s i s

th e p ro b es c o n ta in e d n ic k t r a n s la t e d pAT153 DNA a s w e l l a s

th e c lo n e d HSV DNA fr a g m e n ts , and th e r e fo r e r e v e a le d t h e s e

bands, w h ich were n o t d i s c lo s e d i n th e c o n t r o l s t r i p s .

Homology betw een th e HSV and CMV genomes

S im ila r ex p er im en ts to th o s e a lr e a d y d e s c r ib e d were

c a r r ie d o u t by p ro b in g b lo t s t r i p s c o n ta in in g r e s t r i c t i o n

d i g e s t s o f HSV-1 o r HSV*-2 DNA w ith human c y to m e g a lo v ir u s

(CMV) s t r a in AD169 DNA. The r e v e r s e ex p er im en t o f p ro b in g

b lo t s t r i p s c o n ta in in g CMV Hind I I I fra g m en ts w ith HSV-1 or

HSV-2 DNA was a l s o p erform ed . No h y b r id is a t io n was

d e te c te d under c o n d it io n s w hich a llo w e d EHV-1 and PRV hom ology

to HSV DNA to be d e t e c t e d . However, when e x c e s s probe8( 1 /ug a t a s p e c i f i c a c t i v i t y o f 2 x 10 c .p .m ./y tg ) was u se d ,

and lo n g ex p o su r e s made o f a u to r a d io g r a p h s , some h y b r id is a t io n

was d e te c te d (P ig u re C 2 . l l ) . CMV DNA h y b r id is e d to HSV-2

BamHI £ , and HSV-2 DNA to CMV H in d l l l no and h i ; th e s e r e s u l t s

were rep rod u ced in a seco n d e x p er im e n t. No hom ology betw een

HSV-1 and CMV DNA was d e t e c t e d .

S tu d ie s o f reco m b in a tio n betw een th e HSV-1 and HSV-2 genomes

The tech n iq u e o f marker r e s c u e o f HSV m utants (Stow

and W ilk ie , 1978) h as been v ery s u c c e s s f u l l y u sed to

g e n e r a te HSV-1/HSV-2 i n t e r t y p i c reco m b in a n ts (P r e s to n e t a l . ,

1 78; Stow and W ilk ie , 1978; Marsden e t a l . , 1978; Knipe

- 8 8 -

e t a l . , 1978; C hartrand e t a l . , 1979 and 1 9 8 0 ) . S tr u c tu r e s

o f th e recom b in ant genom es were r o u t in e ly deduced by

com paring t h e i r DNA r e s t r i c t i o n p r o f i l e s w ith th o se o f th e

p a r e n ta l v i r u s e s , and s c o r in g f o r th e p r e s e n c e or a b sen ce o f

r e s t r i c t i o n s i t e s . The ex p er im en ts d e s c r ib e d h ere were aimed

a t th r e e a s p e c t s o f r e c o m b in a tio n . The f i r s t aim was to

i n v e s t i g a t e th e p o s s i b i l i t y th a t o n ly one o f th e fo u r

iso m e r s o f th e HSV genome i s in v o lv e d in r e c o m b in a tio n . The

secon d aim was to d eterm in e w h eth er r e c o m b in a tio n te n d s to

o ccu r in r e g io n s o f h ig h e r in t e r t y p i c h om ology . The th ir d

aim was to a ttem p t t o c o n s tr u c t reco m b in a n ts w ith com p lete

t y p e - s p e c i f i c h e t e r o lo g y betw een r e p e t i t i v e r e g io n s ; i t

was p r e d ic te d th a t such reco m b in a n ts would n o t in v e r t

n o rm a lly in th e s e g m e n t(s ) bounded by h e t e r o t y p ic r e p e t i t i o n s .

T h is a s p e c t i s d e a l t w ith in S e c t io n 4 .

The m u ta tio n i n HSV-1 tsD maps i n TRg/IRg (BamHI q;

P r e s to n , in p r e s s ) , and may c o n v e n ie n t ly be u sed to produce

i n t e r t y p i c reco m b in a n ts p o s s e s s in g HSV-2 DNA in r e p e t i t i v e

r e g io n s . C e l l m on olayers were c o in f e c t e d w ith tsD DNA and

HSV-2 EcoRI bcde (fra g m en ts sp a n n in g th e j o i n t r e g io n )

i s o l a t e d from a g a r o se g e l s , by th e method o f Stow and

W ilk ie ( 1 9 7 6 ) . The m on olayers were in c u b a te d un d er EHu5

a t 3 8 .5 ° , and s i x p la q u e s p ic k ed from ea ch o f f i f t e e n P e t r i

d i s h e s . The n in e ty p la q u es were p u r i f i e d by two f u r th e r

rou n d s o f p la q u e -p ic k in g a t 3 8 .5 ° . V ir u s s to c k s were grown

a t 31° on P e t r i d i s h e s , t i t r a t e d , and in v iv o ^P-DNA was

p r ep a r ed . Genome s t r u c t u r e s were a n a ly se d by r e s t r i c t i o n

en d o n u c le a se d ig e s t io n w ith Xbal , H in d iI I , EcoRI, B g l l l ,

Hpal , Kpnl and BamHI. A l l c r o s s o v e r s mapped w ith in th e

r e s c u in g HSV-2 j o in t fra g m e n ts , and genome s t r u c t u r e s o f

th e s ix i s o l a t e s from any s in g le d is h s u g g e s te d t h a t in

- 89 -

g e n e r a l th e y were n o t r e la t e d .

The re co m b in a tio n e v e n ts w hich o ccu r in an ex p er im en t

o f t h i s ty p e are r a th e r com plex ow ing to th e f a c i l i t y w ith

w h ich seco n d a ry r e c o m b in a tio n e v e n ts occu r in r e p e t i t i v e

r e g io n s . T h is i s d is c u s s e d in d e t a i l in S e c t io n 4 . In

s h o r t , a prim ary c r o s s o v e r in one r e p e t i t i v e r e g io n i s a l s o

p r e s e n t i n th e o th e r r e p e t i t i o n a t th e s ta g e o f genome a n a l y s i s ,

due to seco n d a ry re co m b in a tio n e v e n t s . T h is means t h a t i f th e

p rim ary c r o s s o v e r o ccu rred i n a r e p e t i t i v e r e g io n o f a

segm en t, i t i s , in th e g r e a t m a jo r ity o f c a s e s , n o t p o s s ib le

t o s t a t e th e o r i e n t a t io n o f th a t segm ent d u r in g r e c o m b in a tio n .

N e v e r t h e le s s , r e co m b in a tio n e v e n ts in o r Ug a r e p r e ser v e d

u n am b igu ou sly r e g a r d le s s o f fu r th e r c r o s s o v e r s in r e p e t i t i v e

r e g io n s . T h e r e fo r e , c a r e f u l a n a ly s i s o f th e r e s u l t i n g

genom es from marker r e s c u e o f tsD a l lo w s e i t h e r th e*

o r ie n t a t io n o f b oth L and S a t prim ary rec o m b in a tio n to be

deduced ( c r o s s o v e r s in U- and U g ) , o r th e o r i e n t a t io n o f S

o n ly ( c r o s s o v e r s in TRj/UE^ and U g ) , o r th e o r ie n t a t io n o f

1 o n ly ( c r o s s o v e r in U£ and T R g/IR g), or n e i t h e r o r ie n t a t io n

( c r o s s o v e r s in TRj/lR-^" and TRg/IRg, o r TRg/IRg and Ug, o r

TRs /I K g o n ly ) .

T able C 2.6 shows th e numbers o f reco m b in a n ts

r e p r e s e n t in g each o r i e n t a t io n o f th e r e sc u e d genom e. I t i s

c l e a r from th e s e r e s u l t s t h a t tsD was r e sc u e d w ith th e

genome in b oth th e P and th e 1^ o r i e n t a t io n s , and a l l th e

o b se rv ed c r o s s o v e r s co u ld h ave" occu rred betw een tsD and

HSV-2 EcoRI b or c . I t can n ot be c o n c lu d e d , h ow ever, t h a t

r eco m b in a tio n in th e Ig or Ig^ o r ie n t a t io n s (w ith HSV-2

EcoRI d and e ) d id n o t o c c u r ,' b ecau se th e g r e a t m a jo r ity

o f reco m b in a n ts p o s s e s s e d sy m m etr ica l c r o s s o v e r s in TRg / I H S

and th e r e fo r e m ight e q u a lly w e l l have been th e r e s u l t o f

r e s c u e by HSV-2 EcoRI d or e . The p o s i t io n o f tsD in

- 90 -

TRg/IRg means th a t upon r e sc u e o f t h i s m u ta tio n a t l e a s t one

c r o s s o v e r ev en t in S i s o b l ig a t o r y , w hereas a co n cu rren t

c r o s s o v e r e v e n t in L i s o p t io n a l . I t may be co n clu d ed t h a t

a t l e a s t th e P and 1^ o r ie n t a t io n s o f th e genome p a r t ic ip a t e

in r e c o m b in a tio n .

A secon d f e a t u r e o f reco m b in a tio n i s r e v e a le d by

th e s e r e s u l t s . About te n t im e s a s many reco m b in a n ts

r e s u l t e d from marker r e s c u e o f th e 1^ o r ie n t a t io n compared

w ith th e P o r i e n t a t io n , a lth o u g h EcoRI b and c d i f f e r i n

s i z e by o n ly about 5^ . The d i s t r i b u t i o n o f prim arym

c r o s s o v e r p o s i t i o n s i n a re shown in P ig u re C 2 .1 2 . Two

reco m b in a n ts p o s s e s s e d sy m m etr ica l c r o s s o v e r s in TR / I rL’

so i t was assum ed f o r th e p u rp o ses o f th e diagram th a t th e

prim ary e v e n t co u ld have o ccu rred w ith eq u a l p r o b a b i l i t y

in e i t h e r TR or IH^. R ecom b in ation seem s to have o ccu rred

p r e f e r e n t i a l l y in th e more hom ologous r e g io n s between th e

HSV-1 and HSV-2 genom es.

In a secon d ex p er im e n t, tw e n ty - e ig h t reco m b in a n ts

were i s o l a t e d by marker r e s c u e o f tsD w ith HSV-2 Hindl l l

c d e fg ( c d fg a re th e j o in t fr a g m e n ts ) , o f w h ich i t was

p o s s ib l e to d eterm in e th e s t r u c t u r e s o f tw e n ty -s e v e n .

A l l c r o s s o v e r s in Ug ( 7 /2 7 ) o ccu rred i n r e g io n s o f HSV-1

HRA com plem entary to HSV-2 HindI I I k; o th e r c r o s s o v e r s

were w ith in T R g /I R g , and in a l l e x c e p t one c a s e were

sy m m e tr ic a lly p o s i t io n e d in both r e p e t i t i o n s . O ther

c r o s s o v e r p o s i t i o n s were e i t h e r in TR^/IR^ c l o s e to th e

j o i n t , or in TRg/IRg* th e s e two p o s s i b i l i t i e s co u ld n o t be

d is t in g u is h e d from ea ch o th e r . A lth ough th e r e s c u in g

fra g m en ts ex ten d ed i n t o U^, no c r o s s o v e r s i n were

d e t e c t e d . Prim ary c r o s s o v e r p o s i t i o n s in S from both

- 91 -

ex p er im en ts a re shown in P ig u re C 2 .1 3 . Only HSV-2 EcoRI

j o i n t fra g m en ts produced c r o s s o v e r s a t th e l e f t hand s id e

o f U , and o n ly HSV-2 H in d l l l j o i n t fra g m en ts recom bined a tiD

th e r ig h t hand s i d e . The com p arison o f reco m b in a tio n

f r e q u e n c ie s on th e two s id e s o f Ug was c o n s tr u c te d on th e

assu m p tio n t h a t prim ary r e c o m b in a tio n w ith in T R g /IR g

c o u ld o ccu r w ith e q u a l p r o b a b i l i t y i n e i t h e r ; th e r e fo r e

i n ea ch o f th e two c a s e s th e number o f prim ary re co m b in a tio n

e v e n ts o c c u r r in g in T R g /I R g was d iv id e d by tw o. The r e s u l t s

a re e x p r e s se d a s c r o s s o v e r f r e q u e n c ie s r e l a t i v e to th o se

w ith in HSV-1 BamHI A gain , reco m b in a tio n e v e n ts o ccu rred

p r e f e r e n t i a l l y w ith in th e m ost hom ologous r e g io n s .

- 92 -

JOISCUS5IOH

F e a tu r e s o f n u c le ic a c id h y b r id i s a t io n

H y b r id is a t io n i n s o lu t io n h a s been u sed s u c c e s s f u l l y f o r

many y e a r s to d eterm in e r e la t i o n s h i p s betw een n u c le ic a c id s ,

and th e f e a t u r e s o f t h i s a s s a y h ave been w e l l c h a r a c te r is e d

b oth t h e o r e t i c a l l y and e m p ir ic a l ly . H y b r id is a t io n r a t e i s

m axim al a t a p p r o x im a te ly 2 5 ° below th e h y b r id m e lt in g

tem p era tu re (Tm) , and v a r i e s m in im a lly o v e r a ran ge o f 1 0 -1 5 °

i n t h a t r e g io n . Tm i s r e la t e d to io n c o n c e n tr a t io n and OC

c o n te n t (S c h ild k r a u t and L if s o n , 1 9 6 5 ) , and i s d e c r e a se d by .

a p p r o x im a te ly 1 ° p e r 1 # b a se m ism atch in g (H u tton and Wetmur,

1 9 7 3 ) . The u se o f s in g le stra n d ed DNA bound to a n i t r o c e l l u l o s e

f i l t e r i n th e h y b r id is a t io n r e a c t io n i s a l e s s w e l l -

c h a r a c t e r is e d sy ste m , s in c e under c e r t a in c o n d it io n s th e r a t e

o f r e a c t io n i s l im i t e d by a d i f f u s i o n f a c t o r , and a l s o

c o n c a te n a t io n o f p a r t i a l l y r e a n n e a le d probe to th e f i l t e r - b o u n d

DNA may o ccu r ( F l a v e l l e t a l . , 1 974a and b ) . The u se o f

form am ide i n f i l t e r h y b r id is a t io n s t i l l f u r t h e r r e d u c es th e

p o s s i b i l i t y o f m ea n in g fu l q u a n t i f i c a t io n o f r e s u l t s . In

s o lu t io n h y b r id is a t io n , formam ide r e d u c e s Tm by 0*6° p er 1 $

form am ide, and -the h y b r id is a t io n r a t e by h a l f , a lth o u g h th e

r a t e r e t a i n s an ap p roxim ate in d ep en d en ce o f tem p eratu re in th e

r e g io n o f Tm-2 5 ° (H u tton , 1 9 7 7 ) . Formamide e f f e c t u a l l y

in c r e a s e s th e s t a b i l i t y o f m ism atched h y b r id s on f i l t e r s when

compared w ith what a re presum ably com parable c o n d it io n s i n th e

a b se n c e o f form am ide (Schm eckpeper and S m ith , 1 9 7 2 ) .

T h ere fo re f i l t e r h y b r id is a t io n i n 5 0 # form am ide, w hich0

was th e method u sed in t h i s s tu d y , can p r o v id e an i n d ic a t io n -

o f r e l a t i v e h y b r id is a t io n betw een r e s t r i c t i o n fra g m en ts o f

h e te r o lo g o u s DNAs, but n o t r e l i a b l e q u a n t i t a t iv e e s t im a t e s .

However, q u a n t i t a t iv e e s t im a t io n s o f th e d e g r ee o f hom ology

- 93 -

b etw een h e te r o lo g o u s DNAs d e r iv e d from h y b r id is a t io n

e x p e r im e n ts bear no d i r e c t r e la t io n s h ip to s i m i l a r i t y o f

n u c le o t id e se q u e n c e s , and t h i s i s o f c o u r se th e more

m ea n in g fu l d e s c r ip t io n o f hom ology. For exam p le , i t i s n o t

known w h eth er hom ologous se q u e n c es d e t e c te d i n t h i s s tu d y a re

w id e ly d i s t r ib u t e d w ith in th e hom ologous r e g io n s , or w hether

th e y c o n s i s t o f l o c a l r e g io n s o f v e r y h ig h h om ology . These

a l t e r n a t i v e s co u ld be d is t in g u is h e d by e s t im a t in g th e s t a b i l i t y

o f th e h y b r id s . A la r g e d i f f e r e n c e i n th e e x t e n t o f ap p aren t

hom ology b etw een two s t r a i n s o f v e s i c u l a r s t o m a t i t i s v ir u s

d eterm in ed by th e two m ethods o f h y b r id is a t io n and n u c le o t id e

se q u e n c in g was d is c u s s e d by McGeoch e t a l . ( 1 9 8 0 ) .

In h y b r id is in g h e te r o lo g o u s DNAs, th e Tm o f h y b r id s , and

h en ce t h e ir r a t e o f fo r m a tio n and r e s u l t i n g s t a b i l i t y , i s

d ep en d en t upon th e GC c o n te n t o f hom ologous r e g io n s and th e

e x t e n t o f b ase m ism a tch in g . U sin g th e e q u a t io n o f S c h ild k r a u t

and L if s o n (1 9 6 5 ) and assu m in g t h a t 5 0 # form am ide red u o es Tm

by 30° (H u tton , 1 9 7 7 ) , th e DNAs o f HSV-1, HSV-2, PRV and EHV-1

u nder th e c o n d it io n s u sed have Tm- 2 5 ° v a lu e s o f 4 8 .9 , 4 9 .7 ,

5 1 .4 and 4 4 .4 ° , r e s p e c t i v e l y . T hese a re mean v a lu e s , and Tm

w i l l d i f f e r f o r r e g io n s o f th e genome w ith GC c o n te n ts

d i f f e r e n t from th e mean. In g e n e r a l , h ow ever, h y b r id is a t io n o f

HSV-1 and HSV-2 DNA a t 50° a l lo w s up to 1 0 -1 5 # m ism atch in g ,

45 ° a l lo w s 1 5 -2 0 # , and 40 a l lo w s 2 0 -2 5 # , assu m in g t h a t th e4 4

e f f e c t o f m ism atch in g on Tm i s th e same i n s o lu t io n h y b r id is a t io n

and under th e c o n d it io n s u sed h e r e . H y b r id is a t io n o f HSV-1

and HSV-2 DNA a t t h e s e th r e e tem p era tu res r e s u l t e d i n s im i la r ,

b u t n o t i d e n t i c a l , p a t te r n s (P ig u r e 0 2 . 2 ) . T h is i s i n agreem ent

w ith K ie f f e t a l . (1 9 7 2 ) , who co n clu d ed t h a t th e hom ologous

se q u e n c es p o s s e s s a low d eg ree o f m ism a tch in g . H y b r id is a t io n

o f EHV-1 DNA to HSV-1 o r HSV-2 DNA was much more tem p era tu re -

- 94 -

d ep en d en t in th e 4 0 -5 0 ° range ( f i g u r e C 2 .7 ) , presum ably a s a

r e s u l t o f g r e a t e r m ism a tch in g .

The e q u a t io n o f S c h ild k r a u t and L if s o n (1 9 6 5 ) shows t h a t

hom ologous r e g io n s may v a r y in GC c o n te n t o v e r a 35?* ra n g e ,

o th e r f a c t o r s b e in g e q u iv a le n t , w ith o u t g r e a t ly a f f e c t i n g th e

r a t e o f h y b r id i s a t io n . The d i s t r i b u t i o n o f th e more hom ologous

r e g io n s b etw een HSV-1 DNA and th e o th e r h e r p e s v ir u s DNAs d o es

n o t o b v io u s ly r e f l e c t th e d i s t r i b u t i o n o f G C -rich r e g io n s in

th e HSV-1 genome lo c a t e d by th erm al d e n a tu r a t io n ex p er im en ts

( D e l iu s and C lem en ts , 1 9 7 6 )* I t i s t h e r e f o r e p ro b a b le t h a t th e

r e g io n s o f g r e a t e r hom ology d e t e c te d i n t h i s s tu d y a re l a r g e l y

th e r e s u l t o f g r e a t e r s i m i l a r i t y o f n u c le o t id e se q u e n c e s

r a th e r th a n m erely a r e f l e c t i o n o f l o c a l GC c o n t e n t .

C o l in e a r i t y o f th e genom es o f HSV-1 and HSV-2

H y b r id is a t io n o f c lo n e d DNA fra g m en ts to n i t r o c e l l u l o s e

s t r i p s c o n t a in in g HSY-1 o r HSV-2 r e s t r i c t i o n fra g m en ts showed

t h a t , w i t h in th e r e s o lu t io n a t t a in e d , th e two genom es a re

c o l in e a r ( f ig u r e C2.4» T ab le C 2 * l) . H y b r id is a t io n o f HSV-1

and HSV-2 BamHI fra g m en ts in th e c r o s s b lo t o f f ig u r e C2*6

a l s o d em o n stra ted c o l i n e a r i t y , w ith th e e x c e p t io n o f some

n o n -c o l in e a r hom ology a s d is c u s s e d p r e v io u s ly . E sp arza e t a l .

(1 9 7 6 ) co n c lu d ed t h a t th e r e i s a t l e a s t some d e g r e e o f

c o l i n e a r i t y betw een HSV-1 and HSV-2 DNA from th e o b s e r v a t io n

t h a t h e te r o lo g o u s m utant p a ir s w hich f a i l e d to com plem ent a l s o,V4V-

f a i l e d to reco m b in e . A n a ly se s o f i n t e r t y p io reco m b in a n ts

have shown t h a t c r o s s o v e r s o cc u r th ro u g h o u t th e genome,

r e f l e c t i n g f u n c t io n a l c o l i n e a r i t y . S p e c i f i c f u n c t io n s , su ch

a s th e dPyK gene (H a ll ib u r to n e t a l . , 1 9 8 0 ) , th e DNA p o lym erase

gene (C hartrand e t a l . , 1 9 7 9 ) , and th e IE g e n e s (C lem en ts e t a l . ,

1 9 7 7 ;~ E a sto n and C lem en ts , 1 9 80) a re in e q u iv a le n t l o c a t io n s

in th e two genom es. The m o r p h o lo g ic a l tr a n sfo r m in g r e g io n s o f

- 95 -HSV-1 and HSV-2 a re n o t e q u iv a le n t ly lo c a t e d , but th e DNA in

ea ch r e g io n i s c o l in e a r w ith th e a n a lo g o u s r e g io n i n th e o th e r

genome (R eyes e t a l . , 1 9 7 9 ) . Ruyechan e t a l . (1 9 7 9 ) co n clu d ed

t h a t th e g e n e s s p e c i f y in g gC a r e n o t c o l i n e a r , a lth o u g h t h i s

was n o t su p p o rted by H a ll ib u r to n (1 9 8 0 ) . The HSV-1 BamHI/

HSV-2 BamHI c r o s s b lo t o f F ig u r e C2*6 d id n o t s u g g e s t any

n o n - c o l in e a r i t y i n t h i s r e g io n (0 * 5 3 -0 .6 9 f r a c t i o n a l genome u n i t s ) .

C onserved r e g io n s i n th e genom es o f HSV-1. HSV-2, EHV-1 and FRY

Seven m ajor r e g io n s o f h ig h e r hom ology b etw een HSV-1 and

HSV-2 DNA w ere d em o n stra ted ( I - V I I i n l i g u r e C 2 .1 0 ) , and o f

t h e s e I , ' I I I , V, VI and V II were a l s o hom ologous to PRV and

EHV-1 DNA, a lth o u g h a t a much lo w er l e v e l . The e x t e n t o f

r e g io n ’ l l was more l im i t e d u s in g EHV-1 DNA a s p ro b e , and

r e g io n IV was d e t e c te d w ith PRV a s probe o n ly when HSV-1 DNA

f i l t e r s w ere u se d . A lth ou gh th e same r e g io n s a re con served "

b etw een th e fo u r genom es, th e r e s u l t s d id n o t show w h eth er

th e same DNA se q u e n c e s a r e co n se r v ed in ea ch c a s e . The

o b s e r v a t io n th a t n e a r ly a l l HSV-1 and HSV-2 r e s t r i c t i o n

fra g m en ts showed some l e v e l o f h y b r id is a t io n to th e h e te r o lo g o u s

probe (F ig u r e s C 2.1 and C 2 .2 ) , w h ereas o n ly c e r t a in fra g m en ts

h y b r id is e d to PRV o r EHV-1 p ro b es (F ig u r e C 2 * 7 ), may be

a t t r ib u t e d t o th e much lo w er l e v e l o f h y b r id is a t io n in g e n e r a l

in th e l a t t e r c a s e , and hence a lo w e r l e v e l o f s t r in g e n c y may

be r e q u ir e d to d e t e c t h y b r id is a t io n to th e o th e r fr a g m e n ts .

The sev en r e g io n s o f h ig h e r hom ology p ro b a b ly r e f l e c t c o n s t r a in t s

on DNA se q u e n c es im posed by c o n s e r v a t io n o f amino a c id

se q u e n c e s i n th e p r o t e in s encoded by th e DNA. The f o l lo w in g

p aragrap h s d is c u s s th e i d e n t i t i e s and f u n c t io n s o f p u t a t iv e

co n se r v ed p r o t e in s .

R eg ion I maps w ith in th e HSV-2 m o r p h o lo g ic a l tr a n sfo r m in g

r e g io n (R eyes e t a l . , 1 9 7 9 ) . One p o s s i b i l i t y f o r th e co n serv ed

- 96 -

p r o t e in i s gC, th e s t r u c t u r a l gen e f o r w hich i s lo c a t e d in

t h i s r e g io n but h a s n o t been p r e c i s e l y mapped (M arsden e t a l * ,

1 9 7 8 ; Ruyechan e t a l . , 1 979; H a ll ib u r to n , 1 9 8 0 ? . uyp e-

s p e c i f i c a n t ig e n ic s i t e s have been r e p o r te d f o r t h i s

g ly c o p r o t e in (N o r r ild and V e ste r g a a r d , 1 9 7 9 ) . P erh ap s a more

l i k e l y c a n d id a te i s Vmw IE 1 3 6 * (1 4 3 ) , th e gen e f o r w hich i s

t r a n s c r ib e d i n sm a ll q u a n t i t i e s i n th e p r e s e n c e o f

c y c lo h e x im id e , and i n g r e a t ly in c r e a s e d am ounts a f t e r rem oval

o f th e in h i b i t o r (C lem en ts e t a l * , 1977; J . M cLauchlan and

J .B . C lem en ts , p e r s o n a l co m m u n ica tio n ). The mRNA Jaas a s i z e

o f 5*0 kb and h as been a c c u r a t e ly lo c a t e d on th e genome

w ith in r e g io n I (A nderson e t a l l . 1 9 8 1 ; J . M cLauchlan and J .B .

C lem en ts , p e r s o n a l co m m u n ica tio n ). The f u n c t io n o f th e

p r o t e in i s unknown, a lth o u g h th e HSV-2 f u n c t io n c a u s in g

i n h i b i t i o n o f h o s t m acrom olecu lar s y n t h e s is maps i n th e same

l o c a t io n ( 0 .5 6 - 0 .5 7 ) •

R eg ion I I i s lo c a t e d i n th e p o s i t i o n o f th e s t r u c t u r a l

gen e c o d in g f o r th e HSV DNA p o ly m era se (C hartrand e t a l * , 1 9 7 9 ) .

H a llib u r to n and Andrew ( i9 ? 6 ) were a b le to d e t b c t a d eg ree

o f c r o s s - n e u t r a l i s a t i o n betw een th e DNA p o ly m er a se s o f HSV-1

and HSV-2, but n o t betw een th e s e two v i r u s e s and PRV.

The d a ta o f C on ley .e t a l . (1 9 8 1 ) s u g g e s t th a t r e g io n I I I

c o n t a in s th e gen e c o d in g f o r th e m ajor D N A -binding p r o t e in

(MDBP) o f HSV-1. Yeo e t a l . (1 9 8 1 ) showed t h a t a n t i s e r a to

c e l l s in f e c t e d w ith HSV-1, HSV-2, BMV, EHV-1 and PRV r e a c t

h e t e r o lo g o u s ly p red o m in a n tly w ith MDBP, and th e y p rop osed t h i s

p r o t e in a s a p u ta t iv e h e r p e s v ir u s group s p e c i f i c a n t ig e n .

A lth ou gh MDBP i s l i k e l y to have a c e n t r a l r o l e i n DNA

r e p l i c a t i o n , i t s p r e c i s e fu n c t io n i s unknown. The l o c i f o r

gA and gB a l s o map i n t h i s r e g io n , and t h e s e g ly c o p r o t e in s have

been r e p o r te d to be s e r o l o g i c a l l y co n serv ed i n HSV-1 and HSV-2

(N o r r ild e t a l . , 1978; E b er le and C ou rtn ey , 1 9 8 0 a ) . O th ers

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h ave r e p o r te d t h a t gA and gB p o s s e s s t y p e - s p e c i f i c a n t ig e n ic

s i t e s (P o w e ll and W atson, 1975; C ourtney and P o w e ll, 1 9 7 5 ) .

Yeo e t a l . (1 9 8 1 ) a l s o r e p o r te d l im i t e d c r o s s - r e a c t i v i t y

b etw een th e m ajor c a p s id p r o t e in s (MCP) o f th e f i v e h e r p e s v ir u s e s

th e y c o n s id e r e d . C ourtney and P o w e ll (1 9 7 5 ) showed ty p e -

common a n t ig e n ic s i t e s on th e MCP o f HSV-1 and HSV-2 u s in g

s p e c i f i c a n t i s e r a , and Cohen e t a l , ( 1 9 8 0 b )d em o n stra ted

c o n s e r v a t io n o f MCP i n HSV-1 and HSV-2 by s e r o l o g ic a l and

p r o t e o l y t i c m eth od s. MCP has a m .w t. o f 1 4 -0 ,0 0 0 -1 6 0 ,0 0 0 i n a

w ide ra n g e o f h e r p e s v ir u s e s (S p ea r e t a l * , 1 9 7 8 ) and th e genea - - -

h a s been p r e c i s e l y lo c a t e d f o r HSV-1 (C o sta e t a l . , 1 9 8 1 ) ,^ - - -

c o r r e sp o n d in g w e l l w ith th e p o s i t i o n o f r e g io n V.

R eg ion V II i s c e r t a i n l y l o c a t e d , f o r HSV-1, w ith in th e

c o d in g r e g io n o f th e Vm IE 175 gen e (M -J. M urchie and P*J.

R ix o n , p e r s o n a l c o m m u n ica tio n s). D e t a i le d hom ology s t u d ie s o f

th e j o i n t r e g io n s o f HSV-1 and HSV-2 DNA, a s d is c u s s e d in

S e c t io n 3 , in d ic a t e t h a t hom ology e x te n d s th ro u g h o u t a t l e a s t

th e p o ly p e p t id e co d in g r e g io n o f th e g en e : th e arrangem ent o f

r e s t r i c t i o n s i t e s f a l s e l y g iv e s th e im p r e s s io n t h a t HSV-l/HSV-2

hom ology i n t h i s r e g io n i s c o n f in e d to HSV-1 BamHI y , w hereas

i t d o es ex te n d in t o BamHI k . An IE p o ly p e p t id e th o u g h t to

be a n a lo g o u s to HSV-1 Vmw IE 175 h as been mapped i n a s im i la r

p o s i t i o n i n TRg/IRg o f PRV t(]? o w ell, 1 9 7 9 ) . VBW IE 175

f u n c t io n s i n prom otin g t r a n s c r ip t io n o f e a r ly g e n e s . I t i s

n o te d t h a t , a lth o u g h hom ology betw een HSV-1 and HSV-2 DNA i n

t h i s r e g io n o f th e genome i s g r e a t e r th a n i n a d ja c e n t r e g io n s ,

i t seem s n o t to be a s g r e a t a s in r e g io n s I -V I (P ig u r e C 2 .3 )»

C ourtney and P o w e ll (1 9 7 5 ) r e p o r te d t y p e - s p e c i f i c s i t e s on

t h i s p o ly p e p t id e in HSV-1 and HSV-2, u s in g a s p e c i f i c a n tiseru m

to Vmw IE 1 7 5 .

Knowledge o f th e lo c a t io n s o f gen e f u n c t io n s on th e HSV

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genome i s n o t y e t s u f f i c i e n t l y advanced to a l lo w th e

i d e n t i f i c a t i o n o f th e p r o t e in s w hich may be r e s p o n s ib le f o r

th e g r e a te r hom ology i n r e g io n s IV and V I. W ith th e e x c e p t io n

o f th e p u t a t iv e a n a lo g u e o f Vmw IE 175 i n PRV, n o th in g i s

d i r e c t l y known o f th e p r o t e in s coded by th e hom ologous

r e g io n s i n th e PRV and EHV-1 genom es.

The p r o t e in s d e s c r ib e d above com p rise e a r ly and l a t e ,

s t r u c t u r a l and n o n - s t r u c tu r a l members. I t i s n o ted ;th at th e

dPyK gen e d o e s n o t r e s id e w ith in a r e g io n o f g r e a t e r hom ology

in HSV-1 and liSV -2. The r e g io n s o f l e a s t hom ology betw een

HSV-1 and HSV-2 DNA encode HSV-1 Vmw IE 1-2 and 68 a t th e

TRg/us and IRg/Ug j u n c t io n s , and Vmw IE 110 i n T R i/lR ^.

A s t r i c t com p arison betw een s e r o l o g ic a l and h y b r id is a t io n

s t u d ie s i n th e se a r c h f o r co n serv ed p r o t e in s i s n o t

s tr a ig h t fo r w a r d , s in c e gD h as been r e p o r te d to be co n serv ed

in HSV-1 and HSV-2, b oth s e r o l o g i c a l l y (Sim and W atson, 1973;

H oness and W atson, 1974) and by t r y p t ic ~ p e p t id e mapping

(E ise n b e r g e t a l . , 1 9 8 0 ) . The gen e c o d in g f o r t h i s g ly c o p r o t e in

r e s id e s w ith in Ug (Ruyechan f i l . , 1 9 7 9 ) , none o f th e seq u e n c e s

o f w hich were d e te c te d a s b e in g p a r t ic u la r ly hom ologous in

HSV-1 and HSV-2.

Homology b etw een th e genomes o f HSV and HOMV

A v e r y sm a ll amount o f hom ology b etw een HSV-2 DNA and

a t l e a s t two l o c i in o f th e CMV genom e, and betw een CMV DNA

and TRj/IRjj o f th e HSV-2 genome was d e t e c te d a t th e

tem p era tu re o f h y b r id is a t io n u s e d . I t i s q u e s t io n a b le

w hether su ch a low l e v e l o f h y b r id is a t io n i s m ea n in g fu l a t a l l ,

s in c e i t may r e s u l t from n o n - s p e c i f i c h y b r id is a t io n o f DNA o f

u n u su a l c o m p o s it io n ( e . g . h ig h GC c o n t e n t ) . T hese r e s u l t s

a re i n agreem en t w ith th o se o f Huang and Pagano (1 9 7 4 ) , who

f a i l e d to d e t e c t s i g n i f i c a n t h y b r id is a t io n o f HSV-1 or HSV-2

- 99 -

DNA to CMV DNA. S e v e r a l o th e r l i n e s o f in v e s t i g a t i o n

h ave d em o n stra ted t h a t a p a r t from v ir io n m orphology, g r o s s

genome s t r u c t u r e and some a s p e c t s o f v ir u s grow th , HSV and

CMV show l i t t l e s i m i l a r i t y . For exam ple , CMV d o e s n o t p o s s e s s

a dPyK o r th y m id in e k in a se gen e ( M i l le r e t a l . , 1977;

J e r k o fsk y e t a l . , 1 9 8 0 ) , and e x h i b i t s an IE t r a n s c r ip t io n a l

p a t te r n m arkedly d i f f e r e n t from t h a t o f HSV (Wathen e t a l . ,

1 9 8 1 ) .

D is t r ib u t io n o f r e g io n s hom ologous to HSV DNA i n th e genom es o r " E H V ^ i '" a n a :“F R r “ ----------------------------------: —

The r e s u l t s sum m arised i n l i g u r e C 2.10 show th a t th e '

hom ologous r e g io n s in th e L segm ent o f EHV-1 DNA a re c o l in e a r

w ith th o s e in th e L segm ent o f HSV DNA i n t h e ’ l i , o r i e n t a t io n .

I t m ust be s t r e s s e d th a t th e e x p e r im en ts a llo w e d o n ly

hom ologous r e g io n s to be lo c a t e d . A lth ou gh i t i s r e a s o n a b le to

p ro p o se t h a t o th e r g e n e s a r e c o l in e a r i n th e two v ir u s genom es,

th e r e i s no e v id e n c e to su p p o rt t h i s h y p o t h e s is . Some hom ology

was d e t e c te d w hich d o e s n o t map c o l i n e a r l y . HSV-2 H in d l l l e

h y b r id is e d to some e x t e n t to th e same r e g io n &s HSV-2 Hindi H h .

T h is m igh t r e p r e s e n t a l im i t e d r e g io n o f g e n e t ic n o n - c o l in e a r i t y ,

o r m ight be an a b er ra n t r e s u l t i n t h a t i t d o e s n o t s i g n i f y

f u n c t io n a l c o l i n e a r i t y . A p p a ren tly a b e r r a n t h y b r id is a t io n o f

HSV ( e s p e c i a l l y HSV-2) fra g m en ts c o n ta in in g th e S term in u s to

th e m id d le o f L i n EHV-1 i s d is c u s s e d b e lo w .

The c o n c lu s io n s f o r PRV a r e b ro a d ly i n agreem ent w ith

th o s e o f Rand and B en -P o ra t (1 9 8 0 ) , who d e s c r ib e d th e p a t te r n

o f h y b r id is a t io n betw een th e HSV- and PRY genom es u s in g v i r io n

DNA, n o t in g t h a t m ost o f th e hom ologous se q u e n c e s a re s i t u a t e d

in th e L segm en t. The p a t te r n o f hom ologous r e g io n s in th e

L segm ent o f PRV DNA shown in F ig u re C2*10 d o e s n o t f i t a

c o l in e a r arrangem ent w ith r e s p e c t to HSV DNA. I n v e r s io n o f th e

r e g io n h y b r id is in g to HSV-2 H in d iII h - e - g would le a d to

- 100 -

ap p rox im ate c o l i n e a r i t y betw een th e hom ologous r e g io n s in th e

1 segm ent o f PRY DNA and th e I^ o r i e n t a t io n o f HSY DNA

( i . e . a n a lo g o u s to th e c o l i n e a r i t y o f EHV-1 and HSV DNA).

T h ere fo re th e p o s s i b i l i t y e x i s t s t h a t t h i s r e g io n o f th e PRV

genome i s in v e r t e d w ith r e s p e c t to HSV. The a c t u a l s i t u a t io n

may b e , o f c o u r s e , more com p lex . As w ith EHV-1 DNA, fra g m en ts

from HSV TRg/lR g h y b r id is e d to e q u iv a le n t p o s i t i o n s in PRV DNA,

and th e o r i e n t a t io n o f HSV-1 BamHI k and £ w ith r e s p e c t to ih©

PRV genome i s in agreement with the o r ien ta tio n o f the PRV gene

analogous to HSV-1 Vmw IE 175 (Pow ell, 1979; P.J. Rixon,

P e r so n a l co m m u n ica tio n ).

N o n -c o lin e a r hom ology

N o n -c o lin e a r hom ology was d e te c te d w it h in th e HSV-1

genome and betw een HSV-1 and HSV-2 DNA u s in g th e c r o s s b lo t

t e c h n iq u e . Such hom ology was n o t d e te c te d in ex p er im en ts

where c lo n e d DNA p ro b es and n i t r o c e l l u l o s e s t r i p s were u s e d .

The d i f f e r e n c e i s p ro b a b ly a r e s u l t o f th e d i f f e r e n t t e c h n iq u e s .

The c r o s s b lo t method a l lo w s th e d e t e c t io n o f r e g io n s o f poor

hom ology s in c e a t t h a t p o in t i n th e n i t r o c e l l u l o s e f i l t e r th e r e

i 3 no c o m p e t it io n f o r th e probe by f i l t e r - b o u n d seq u e n c es o f

g r e a t e r hom ology. The s ig n i f i c a n c e o f n o n -c o l in e a r hom ology

i s i n d o u b t. M ost in s t a n c e s in v o lv e th e in v e r t e d r e p e a t s o r

t h e i r ju n c t io n s w ith u n iq u e r e g io n s , s e q u e n c e s known to p o s s e s s

r e g io n s o f i n t r a - o r i n t e r - s t r a i n s i z e v a r i a b i l i t y and, a t

l e a s t i n some in s t a n c e s , to c o n ta in m u lt ip le r e i t e r a t i o n s o f

s h o r t G C -rich seq u e n c e s (L o n sd a le e t a l . , 1 9 8 0 ; S e c t io n p o f

R e s u lt s ; M -J. M urchie and P .J . R ixon , p e r s o n a l c o m m u n ica tio n s).

I t i s p o s s i b l e , t h e r e f o r e , t h a t n o n -c o lin e a r hom ology r e f l e c t s

th e l o c a t io n s o f su ch se q u e n c e s w ith in th e genom e. A s im i la r

c o n s id e r a t io n may be a p p lie d to th e o b serv ed h y b r id is a t io n o f

HSV fra g m e n ts c o n ta in in g th e S term in u s to r e g io n s in th e L

- 101 -

segm ent o f b oth PRY and EHV-1 DNA, and o f s e v e r a l HSV fra g m en ts

to r e p e t i t i v e r e g io n s o f th e PRV and EHV-1 genom es (T a b le s* ^ *

C 2 .4 -C 2 .5 ) . The n o n -c o lin e a r hom ology in t h e s e c a s e s was f a r

more p rom in en t th an t h a t d e t e c te d w ith in HSV-1 DNA or betw een*

th e HSV-1 and HSV-2 genom es, p ro b a b ly a s a r e s u l t o f th e

g e n e r a l ly low l e v e l o f h y b r id is a t io n o f PRV and EHV-1 DNA to

HSV DNA.

G e n e tic r e la t e d n e s s o f h e r p e s v ir u s e s

The main approach to s t u d ie s o f g e n e t ic r e la t e d n e s s among

h e r p e s v ir u s e s h as been an im m u n o lo g ica l o n e . A number o f

t e c h n iq u e s a re w id e ly u se d , in c lu d in g v ir u s n e u t r a l i s a t i o n ,

immune p r e c i p i t a t i o n and im m u n o flu o rescen ce . A n t is e r a may be

r a is e d a g a in s t v i r i o n s , n u c le o c a p s id s , v i r u s - i n f e c t e d c e l l s o r

p u r i f i e d p r o t e in s . The c r i t e r i o n o f r e la t e d n e s s i s w hether

an a n tiseru m produced a g a in s t one v ir u s c r o s s - r e a c t s w ith

a n o th e r v ir u s i n th e t e s t u s e d . The p e r t in e n t p o in t h as been

made, h ow ever, t h a t a n t ig e n s w ith t y p e - s p e c i f i c d e te r m in a n ts

a l s o p o s s e s s type-com mon s i t e s , th u s l i m i t i n g th e c o n c lu s io n s

w h ich may be drawn by t h i s ap proach to h e r p e s v ir u s

r e la t e d n e s s ( K i l l in g t o n e t a l . , 1 9 7 8 ; Yeo e t § 1 . , 1 9 8 1 ) .

The s e a r c h f o r a h e r p e s v ir u s group s p e c i f i c a n t ig e n has

n o n e t h e le s s p roceed ed from th e MCP (Kirkwood e t a l . , 1972;

H oness e t a l . , 1 9 7 4 ) to th e v i r i o n g ly c o p r o t e in s (N o r r ild e t a l . ,

1 9 7 8 ) and a t p r e s e n t c e n t r e s on th e MDHP (Yeo e t a l . , 1 9 8 1 ) .

K il l in g t o n e t a l . (1 9 7 8 ) have p rop osed an im m u n o lo g ica l

c l a s s i f i c a t i o n o f h e r p e s v ir u s e s w ith two m ain c l a s s e s :

a n e u tr o s e r o n , c o n ta in in g v ir u s e s w hich c r o s s - n e u t r a l i s e , and

a s e r o n , c o n ta in in g v ir u s e s w hich c r o s s - r e a c t a n t ig e n i c a l l y

but w hich do n o t n e c e s s a r i ly c r o s s - n e u t r a l i s e . T h is group h as

p r e se n te d e v id e n c e , from p erh ap s th e c l e a r e s t work on th e

s u b j e c t so f a r , th a t HSV-1, HSV-2 and EMV form a n e u tr o se r o n ,

- 102 -

and t h a t th e s e th r e e v i r u s e s p lu s PR? and EHV-1 form a se ro n

( K i l l in g t o n e t a l . , 1977 aad 1978; Yeo e t a l . , 1 9 8 1 ) . The

im p l ic a t io n th a t HSV-2 i s more c l o s e l y r e la t e d to HSV-1 th a n i s

EMV, and t h a t PRV and EHV-1 a re more d i s t a n t l y r e la t e d t o HSV-1,

has th e su p p o rt o f n u c le ic a c id h y b r id is a t io n s t u d ie s (R L eff

e t a l . , 1 9 72; Bronson e t a l . , 1 9 7 2 ; S te r z e t a l . , 1 9 7 4 ) . :

N u c le ic a c id h y b r id is a t io n le n d s i t s e l f a s an a l t e r n a t iv e

ap p roach to th e s tu d y o f g e n e t ic r e la t e d n e s s betw een h e r p e s v ir u s e s .

In th e p a s t , how ever, t h i s te c h n iq u e h as been u n s a t i s f a c t o r y

i n t h a t o n ly g r o s s genom e-to-genom e hom ology was m easured .

M oreover, m ost h e r p e s v ir u s DNAs ch are l e s s th an 5 -1 0 # hom ology,

a l e v e l w h ich by t h i s method i s d i f f i c u l t to d i s t i n g u i s h from

z e r o . The a v a i l a b i l i t y o f c lo n e d DNA fra g m en ts and th e u se o f. .

b lo t h y b r id is a t io n c l e a r l y en h an ces th e u s e f u ln e s s o f

h y b r id is a t io n , a s d em on strated i n t h i s s tu d y . L iq u id

h y b r id is a t io n e x p er im en ts would a l lo w b e t t e r q u a n t i f i c a t io n o f

r e s u l t s , a lth o u g h n u c le o s id e se q u e n c in g o f a n a lo g o u s g e n e s i n

d i f f e r e n t h e r p e s v ir u s genom es w i l l su p p ly th e u l t im a te

e s t im a t io n o f hom ology.

One a p p l i c a t io n o f th e s tu d y p r e s e n te d h er e i s t h a t th e

l o c a t io n s o f EHV-1 g e n e s can be p r e d ic t e d by v i r t u e o f th e

c o l i n e a r i t y betw een hom ologous r e g io n s i n EHY-1 and HSV DNA.

F u r th e r , an unmapped gen e i n e i t h e r EHV-1 o r PRV DNA c o u ld ‘ be

lo c a t e d by u s in g a s a probe th e HSV-1 g e n e , p r o v id in g th e r e i s

s u f f i c i e n t hom ology betw een th e r e s p e c t iv e DNA s e q u e n c e s . For

exam p le , th e MCP gene i n EHV-1 and PRV c o u ld be lo c a t e d u s in g

an HSV-1 MCP gene p ro b e . T h is would o b v ia te th e need to l o c a t e

t h i s f u n c t io n by marker r e s c u e o f a m u ta tio n i n th e g en e , w hich

r e q u ir e s a m utant, o r by a n a ly s i s o f i n t e r t y p i c reco m b in a n ts ,

w hich r e q u ir e s two s e r o ty p e s w ith d i s t i n c t i v e r e s t r i c t i o n and

p o ly p e p t id e p r o f i l e s w hich are a b le to reco m b in e . I t may

p rove p o s s ib l e to l o c a t e w ith HSV-1 p ro b es even a p p a r e n t ly

- 103 -

n on -h om ologou s g e n e s i n PRV o r EHV-1 DNA, su ch a s th e th ym id in e

k in a se g e n e , by d e c r e a s in g th e s tr in g e n c y o f h y b r id is a t io n .

Homolo/ry and re co m b in a tio n i n HSV

I t was co n c lu d ed from th e s t r u c t u r e s o f HSV-l/HSV-2

in t e r t y p i c reco m b in a n ts produced by m arker r e s c u e o f HSV-1 tsD

t h a t r e c o m b in a tio n te n d s to o ccu r in r e g io n s o f g r e a te r

h om ology. In a t te m p tin g to o b ta in th e d i s t r i b u t i o n o f c r o s s o v e r s

betw een th e two ends o f L and S i t was assum ed , and p rob ab ly

n o t u n r e a so n a b ly , t h a t re co m b in a tio n w ith in e i t h e r TRg or IRg

and e i t h e r TR or IR^ o c c u r s w ith eq u a l p r o b a b i l i t y w ith th e

a p p r o p r ia te s&gment i n e i t h e r o r i e n t a t i o n . Even i f t h i s were

n o t th e c a s e , th e c o n c lu s io n would be u n a f f e c t e d . Morse e t a l .

(1977 ) s u g g e s te d from t h e i r l im i t e d s tu d y t h a t c r o s s o v e r s

ten d ed to c l u s t e r i n th e r e g io n o f 0 .4 and 0 .6 2 - 0 .6 9 f r a c t i o n a l

genome u n i t s . An a n a ly s i s o f reco m b in a n ts from s e v e r a l

l a b o r a t o r ie s rev iew ed "by H a ll ib u r to n (1 9 8 0 ) show s a r a th e r good

f i t betw een th e d i s t r i b u t i o n o f c r o s s o v e r s th ro u g h o u t th e

genome and th e d i s t r i b u t i o n o f hom ology p r e se n te d h e r e , w ith th e

e x c e p t io n o f a h ig h fre q u en cy o f c r o s s o v e r s a t 0 .6 2 - 0 .6 9

o b se rv e d by Morse e t a l . (1 9 7 7 a ) but n o t by o th e r g ro u p s.- - - ^ ^

No reco m b in a n t h as y e t been d e s c r ib e d , in c lu d in g th o se

a n a ly se d h e r e , w hich h as a c r o s s o v e r in th e r e g io n o f l e a s t

hom ology betw een th e two genom es, t h a t i s , a t th e TRS/U g and

IRg/Us j u n c t io n s .

Do e s o n ly one HSV genome arrangem ent p a r t ic ip a t e i n reco m b in a tio n ?

The r e s u l t s o f th e work p r e se n te d h ere a re im p o rta n t

w ith r e s p e c t to th e h y p o th e s is t h a t n o t a l l f o u r genome

arran gem en ts may p a r t ic ip a t e i n th e g e n e r a t io n o f v ia b le

in t e r t y p i c rec o m b in a n ts . A n a ly se s o f th e numbers o f c r o s s o v e r s

in i n t e r t y p i c reco m b in a n ts have su g g e s te d t h a t o n ly one o r both

o f th e P and Ig arran gem en ts p a r t ic ip a t e s in reco m b in a tio n

- 104 -

(M orse e t a l . , 1977 ; P r e s to n e t a l . , 1 9 7 8 ; Roizman e t a l . ,

19 7 9 ; W ilk ie e t a l . , 1 9 7 9 b ) . The e v id e n c e i s t h a t genomes

p o s s e s s in g an odd number o f c r o s s o v e r s in UL d is p la y th e l e a s t

number o f c r o s s o v e r s i n th e P o r I g a rra n g em en ts . In d eed ,

t h i s h y p o th e s is f in d s su p p ort i n th e o b s e r v a t io n th a t th e

m a jo r ity o f v i r i o n DNA o f reco m b in a n ts B x l ( 2 8 - l ) and RE4

i s i n th e P o r I g , and I g , a rra n g em en ts , r e s p e c t i v e l y

(P r e s to n e t a l . , 1978; S e c t io n 4 o f R e s u l t s ) . The argum ent

h as th r e e w ea k n esses w hich were em p h asised by i t s p r o p o se r s .

F i r s t l y , i t i s based on a sm a ll number o f reco m b in a n ts w ith an

odd number o f c r o s s o v e r s i n U^: 9 /6 2 reco m b in a n ts produced from

g e n e t ic c r o s s e s in th e s tu d ie d o f Morse e t a l . (1977 .) ,

P r e s to n e t a l . (1 9 7 8 ) , Marsden e t aJL. (1 9 7 8 ) and H a llib u r to n

e t a l . ( 1 9 8 0 ) . The a ssu m p tio n t h a t an^odd number (n ) o f- - • ~ tK * rs

r e c o m b in a tio n e v e n ts i n th e genome i s more l i k e l y to o ccu r

than an even number ( n - f l ) m ust be v iew ed w ith c a u t io n s in c e

th e m a jo r ity o f reco m b in a n ts ( 44/ 6 2 ) p o s s e s s even numbers o f

c r o s s o v e r s i n th e genom e. S e c o n d ly , u n d e te c te d c r o s s o v e r s i n

th e reco m b in a n ts w ith odd numbers o f c r o s s o v e r s i n co u ld

red u ce th e number p u t forw ard a s e v id e n c e . At l e a s t two h^ve been

shown to have an a d d i t io n a l c r o s s o v e r n ea r th e 1 term in u s

( S e c t io n 4 o f R e s u l t s ) . 'T h ir d ly , th e way in w hich reco m b in a n ts

were made may have b ia se d t h e ir o r ie n t a t io n d u rin g

r e c o m b in a tio n . R ecom b in ation i s h o m o lo g y -d ep en d en t, and

th e r e fo r e recom b in ant genome s t r u c t u r e s a r e d ic t a t e d n o t o n ly

by th e p a r t ic u la r m utants u sed i n a g e n e t ic c r o s s and by th e

seq u e n c e s a v a i la b le f o r r e c o m b in a tio n i n th e c a s e o f

marker r e s c u e , but a l s o by th e e x t e n t and d i s t r i b u t i o n o f

hom ology w ith in th o s e se q u e n c e s .

The secon d argum ent in fa v o u r o f th e h y p o th e s is t h a t

no t a l l fo u r arran gem en ts ta k e p a r t in rec o m b in a tio n stem s

- 105 -

from g e n e t ic m apping o f m utant m ark ers. C lem en ts e t a l . (1 9 7 6 )

have made th e p o in t t h a t in v e r s io n o f 1 and S would be

e x p e c te d to le a d to eq u a l rec o m b in a tio n f r e q u e n c ie s betw een

any m arker in L and any marker i n S . That i s , i t would n o t be

p o s s ib l e to a l i g n m arkers i n L w ith r e s p e c t to th o s e i n S .

Brown a t a l . (1 9 7 3 ) o b ta in e d a l i n e a r g e n e t ic map betw een

s e v e r a l m arkers i n and one i n T R g/lR g. U n fo r tu n a te ly ,

Stow e t a l . (1 978) were u n a b le to c o r r e la t e t h i s map w ith th e

p h y s i c a l ly d eterm in ed l o c a t io n s o f th e m ark ers, and t h e r e fo r e i t

was n o t p o s s ib l e to s t a t e w hich o r ie n t a t io n o f th e g e n e t ic

map r e p r e s e n t s . P a r r is e t a l . (1 9 8 0 ) c la im e d t o ” be a b le to

c o r r e la t e th e g e n e t i c a l l y and p h y s i c a l ly d eterm in ed mapping

p o s i t i o n s o f s e v e r a l m u ta t io n s . T h e ir in t e r p r e t a t io n was th a t

m arkers i n L and S were, n o t e q u id is t a n t b eca u se o n ly one o r

b oth o f th e I L and Ig-^ arran gem en ts p a r t i c ip a t e s in g e n e t ic

re c o m b in a tio n ; C le a r ly t h i s i s th e o p p o s it e c o n c lu s io n from

th a t a t t a in e d by argum ent from recom b in an t genome s t r u c t u r e s .

The d a ta p r e se n te d h e r e show t h a t a t l e a s t th e P and 1^

arran gem en ts a re a b le to p a r t ic ip a t e i n t h e g e n e r a t io n o f

v i a b l e r e co m b in a n ts , but t h a t a v a i la b le hom ologous seq u e n c es

p r o fo u n d ly a f f e c t th e fr e q u e n c y o f re co m b in a tio n i n each

o r ie n t a t i o n . Taken t o g e th e r w ith th e above c o n tr a d ic to r y

c o n c lu s io n s , th e r e i s a t p r e s e n t no good r e a so n f o r su p p o sin g

t h a t n o t a l l fo u r arran gem en ts a re a b le to reco m b in e . A

r e c e n t c o n t r ib u t io n i s th e h y p o th e s is o f H oness e t a l . ( 1 9 8 0 ) ,

who p r e s e n te d a d e t a i l e d g e n e t ic a n a ly s i s o f s e v e r a l HSV-1

m ark ers, and co n clu d ed t h a t rec o m b in a tio n i n HSV o c c u r s by a

c i r c u la r o r co n ca te m e r ic m echanism . T h is c o n c lu s io n i s n o t

n eg a ted by in c lu s io n i n th e s tu d y o f HSV-1 t s c 7 5 , w hich had

p r e v io u s ly been e r r o n e o u s ly mapped in each a seq u en ce (K hipe

2-k 9 1 9 7 9 ) , but has s in c e been shown to map o n ly in th e

- 106 -

c se q u e n c e s (P r e s to n , i n p r e s s ) . Two f e a t u r e s o f t h i s m odel

a r e t h a t i t b e t t e r accom m odates i n c l u s i o n o f a l l fo u r

arra n g em en ts i n r e c o m b in a tio n , and t h a t a l l reco m b in a n ts

c o n c e p tu a lly have an even number o f c r o s s o v e r s .

Table 0 2 .1

H y b r id is a t io n o f n ic k t r a n s la t e d recom b in ant p lasm id

c o n t a in in g HSV i)HA fra g m en ts to n i t r o c e l l u l o s e b lo t

s t r i p s o f HSV-1 and HSV-2 BamH I d i g e s t s .

The d a ta a re ta k en from F ig u re C2 A .

TableC2.1

c lo n e d fragm en t probe

HSV-2 PHA BamH I s t r i p s

HSV-1 DNA flamh I . s t r i p s

HSV-2 Hind I I I a ab e f k m s t vw f h i 1 o c !f ! g ' H1

HSV-2 Hind I I I b c d h i j q r x i* a c e m a' f 'g *

HSV-2 Hind I I I e ab M j m o yz h' j 1 d h r w b 1 j 1

HSV-2 Hind I I I h ab n s t k ! l* g pq uv d ’ e 1 i 1

HSV-2 Hind I I I 1 1 c 1d f e 1 f* j n

HSV-2 Hind I I I n r s t

HSV-2 Hind I I I o f b

HSV-2 Hind I I I 12 g 1 u yz a 1 b' j k pq x y zc ‘d* g 1 k ' l 1 m1

HSV-1 BamH I k g u v k pq s t

HSV-1 BamH I y g* y

HSV-2 BamH I g g u v k pq s t

Table C2.2

H y b r id is a t io n ol' n ic k t r a n s la t e d EHV-1 and PRV

HRA to n i t r o c e l l u l o s e b lo t s t r i p s o f th e HSV-1

BamH I d i g e s t . B ata are tak en from F ig u re C 2 .7 .

BamH I fra g m en ts to which the p rob es h y b r id is e d ar

in d ic a t e d , th e number o f c i r c l e s d i s p la y in g an

e s t im a t io n o f th e amount o f h y b r id is a t io n .

Table 02«2

llSV-1 Haaui I 50° 45° 45° -£* o c

i'r agmen t EHV-1 PRV EIIV-1 EHV-1

ci 00 00 000bc 00 00 000aefiS 000 00hij _ic,k ooo 00 000 001m 00 00n0 00 00Pa 000 00 000 00r 000 00stuv 00wX

y 0 00 00 00za' 000 00b’ 00c 1d.1 0e!T’g 1a 1i fj ’

•

Table C2.5

H y b r id is a t io n o f n ic k t r a n s la t e d EHV-1 and PRV

HislA to n itr o c e l- j -u lo s e b lo t s t r i p s o f th e HSV-2

BamH I d i g e s t . D ata a re tak en from F ig u re C 2 .7 .

BamH I fra g m en ts to w hich th e p ro b es h y b r id ise d

a re in d ic a t e d , th e number o f c i r c l e s d i s p la y in g

an e s t im a t io n o f th e amount o f h y b r id is a t io n .

The u se o f th e H i n d l l l / BcoHI d i g e s t o f HSV-2 DNA

in a n o th e r ex p er im en t con firm ed th a t h y b r id is a t io n

was to HSV-2 BamHI t and n o t a*

Table U2.3

HSV-2 Bamli I fragm en t

50°EHV-1

45°PRV

45°EHV-1

40°EilV-1

a

i1

b 000 0 0c 00 00 000a 000 0e 0f 0 0 0&‘>g 000 00 000 00ni;J 000 000k1m 0n0 00 0P 0 0a • 0r3 t 00 0U 000 000 000 00vwX oyza ’D*c ’ d fe*f '• i6 00 00

hi 000 00i !j* * 000k i i*m’ .

L_ . . . . . . .

00

Table C2.4

H y b r id is a t io n o f n ic k t r a n s la t e d recom binant

p la sm id 3 c o n ta in in g HSV DNA fra g m en ts to n i t r o c e l l u l o s e

b l o t ’s t i i p c o f EHV-1 DNA d i g e s t s .

D ata f o r B g l l l and B g l l l / EooRI d i g e s t s are

ta k en from F ig u r e C2*8. Fragm ents i n p a r e n th e se s

h y b r id is e d more w ea k ly . The tem p eratu re o f h y b r id is a t io n

was 40° e x c e p t f o r EHV-1 BamHI , when i t was 4 5 ° . In th e

l a t t e r ex p er im en t th e in c r e a s e d tem p era tu re s i g n i f i c a n t l y

red u ced h y b r id is a t io n when HSY-2 H ind l l l a , b and h were

u sed a s p r o b e s .

Tabl

e C

ZA

IIto«H

0

©co

•© 8 88 ■p

O1o* ©Cl 0 00 .o ,o

•p03

< p51IS> 3

8 %e. 0 0 0 0

Bs g,

cd ghi *8 *8 ■8 ■8PS•H

ft -8 i.o

1 0&0 0

.©0

&up

■< to

S SSi

/ » \0

' r f 'NwX

c•H

f t

y~\0

o

Pi

f t . o■ o 0 .o 1 1 1 0 0

.©0

.on5

&+» c (0

g £cc> o

Vt0

■8 n—«*

'iov—*B

Vi00 0

%V

Vi0

V0

■80

? *8.© •8 .©

0 ,© 0 0

M

X

r O | 0 1 j e l

M «—* ML J wt jMM

P iM

P IH

' S i d |• g .

OJ CJI I> >

XI(ViI>a a a a

Table C2.5

H y b r id is a t io n o l‘ n ic k t r a n s la t e d recom binant

p la sm id s c o n ta in in g HSV DNA fra g m en ts to

n i t r o c e l l u l o s e b lo t s t r i p s o f PHV d i g e s t s .

D ata are tanen from F ig u re C 2 .9 . Fragm ents in

p a r e n th e se s snowed l e s s h y b r id is a t io n .

Table C2.H

c lo n e d fragm en t p ro be

PRV DMA BamH I s t r i p s

PRV HR A Kpn I s t r i p s

HSV-2 Hind I I I a t> (1 1 ) (o ) a (d )

HSV-2 Hina I I I b c d g c i gh rn

HSV-2 Hind 111 e a (o ) d

HSV-2 Hind I I I h a k c jk

HSV-2 Hind I I I 1 - -

HSV-2 liinct I I I n - -

HSV-2 Hind I I I 0 - -

HSV-2 Hind I I I 2£ i j k o ( a ) (b )

g S i !ef)e HHJk (a )u ; (d) u ) ( i ) -

(b)

llS V -l BamH I k ±1 k 0 (a ) (b )

i l i S i ' 10"

e jk (a )ic 7 (d ) ( i )(1 )

(b )

HSV-1 Hamll i 1 i t e gh

HSV-2 Barnll I & i j k o (a ) (b )

S i S i ' l ° "

e » (a )u ; (a ) u )U ) ■

(b )

Table C2 .6

Genome arran gem en ts o f recom b in an ts produced by

m arker r e s c u e o f 1ISV-1 ts l) w ith HSV-2 EcoR I

j o i n t fr a g m e n ts . The o r ig in a l arran gem en ts o f the

t s l) genome in v o lv e d in reco m b in a tio n were deduced

from th e r e s u l t i n g recom binant s t r u c t u r e s , a c c o r d in

to argum ents d e s c r ib e d in th e t e x t , and a re l i s t e d

w ith th e numbers o f recom b in an ts r e f l e c t i n g

each p a r t ic u la r arrangm ent.

Tabl

e 02

o

COp

<H So a

•HUrOo) arQ Oa o3 O KN 03a u i—1 o i—1 o CM rH O O r - H-

73 03a aM Hi

03 0303 03 03 03 03 'V* wPJu _M

aCD CD CD 'H £h

a"v. \ 1 a a a 73 7j 0to 03 03 q q q rH rH aFi m CQ 03 03 ca Pi 3 3 3 3 a •H0 to CD CD CD EH EH a> h-H . hH 03 a 03 ao a a 1=2 a 'a a « ca cd vd a 0CO q S3 a a q a M M M M M pto 3 3 3 3 3 N ' \ : — • 0o H • iH 03 i a 73 aFi 1h-H . t-H i hI hH .ca cd a a ao CD CD CD CD C3 CD EH EH EH EH EH

---------

olCM P fHJ ^ Ot=> 0 acn a o l a l Ol 0l Ol a l 0rd is a

aj Fi Fi Fh u u a •ri50 Fhf*4 ft_| O O o o o o at .£J HW

•H Ol 0 l 431 a l Ol 43| 431 a l 431 o lH0

3 H po ca Fh 0to ol o a0 o piFh f^l a l

a ao 0•H •H

'H a•H •H 0O O a0 0 •H

• f t f t da to a 0 a Fh

a a o d 0 Si 0 0 0a o o •H 3 •H 3 •H P 0o •H •H P <h <H 0 Fh

0 •H p P a 03 •H i *ri a aa p cd C4 p O O po Cti p P a •t 0 •V 0 a oa P a a 0 a ft a f t o a0 a 0 0 *H o CO o 0 •H FhCjQ 0 •H •H Fi •rH rt •H Si P p

•H fh a O P 3 P 3 a 0«H u o o cd a pO o »-h P 03 p hH , a a

03 h=\ i 03 a a 0 0a f t M M M 0 •* 0 *v a Po •rH a a a Fh 0•H a a a a in 0 Fh 0 o FhP •H •H •H ♦H O p o p ftcd Fh Fh Fh Fhp 0 0 0 0 ft 0 ft 0 0 0d a a a a > > s i P0 o o o o a a a a p a■H a a a a *H •H •H •H •H •HFh 0 0 0 0 0O w 50 50 50 it-H . 03 03 a

oCP>

3EH

I

Figure C2.1

HSV-l/HSV-2 i n t e r t y p i c hom ology. R e s t r ic t io n d ig e s t

o f HSV-1 or HSV-2 D M , in d ic a te d a t th e top o f each

p a ir o f t r a c k s , were t r a n s fe r r e d from th e same g e l

t o n i t r o c e l l u l o s e . The b lo t s t r i p s were h y b r id ise d

w ith n ic k t r a n s la t e d HSV-1 (1 ) or HSV-2 (2 ) DNA, and

a u to r a d io g r a p h e d . The f i v e p a ir s o f tr a c k s on the

l e f t r e p r e s e n t 0 .5 ^ (upper p a n e ls ) and 1 f* a garose4 ^ 4

g e l s ( lo w e r p a n e l s ) . The f i v e p a ir s o f tr a c k s on

th e r ig h t r e p r e s e n t 1 # a g a ro se g e l s , th e low er4

p a n e ls show ing a lo n g e r exp osure o f th e bottom

p a r t o f th e g e l . Tracks o f a p a ir were n o t

a u to ra d io g ra p h ed f o r eq u a l d u r a t io n s . The

tem p era tu re o f h y b r id is a t io n was 4 5 ° .

C/5 9" CM X ^

CM “! > - C/5 CT _ , X CO ^

5 E ^s i -

*■ 0-J*£ o CT « ♦*a

d u ^ M l c i rM R A

T - c o a « t

a *-

« i i l l • I I I ' _ Jk w m » <

rco<u.c :=> —c og-w </i*-M i l 1

> J">N ■« x u i v o f : . -

5 c Nc/5 a X * i -

o "X T -

3 W>0- T-

_ II E CM

>CO m - I

c "a* i-

“ CM<0aX T-

■ M l 1 M l■ M l 1 M

19 uI I f il .

CO -u u u 1) H-C-—.05 ■* - £ 0 £ « - 3E S * 5 > « J o TJ *■ a , j c

X 0) -C

M i l M i l M 1 0 1H H M i l 11 H I M

i u u *o a> t ■fljk

lm

n o Pq

rst u X N -jg -y '0» i - X ' - — *°

* x - o D) c" E

M ■ 1 « « «M M l III

RJ X -Q >*-X • - o « Ol

_ j* —_ C 0 c > - tn«-E a § J X>^, <0 UTOOJ.O^C —

■ M■ M B I"O 0 5 * y —J?S-CD

£ o a crw^ 3 2 . eg -O «

■ •~ - _____ _ ____ " ii -■m m

• l II Idl 00

CD

F ig u r e C 2.2

LEFT HAND FIVE PAIRS OF TRACKS

1ISV-1/HSV-2 i n t e r t y p i c hom ology. R e s t r i c t io n d i g e s t s

o f HSV-2 DNA, in d ic a te d a t th e to p o f each p a ir o f

t r a c k s , were tr a n s fe r r e d from a 0 .5 /2 a g a r o se 0 e l so4

n i t r o c e l l u l o s e . The b lo t s t r i p s were h y b r id is e d

w ith n ic k t r a n s la t e d HSV-1 (1 ) o r HSV-2 (2 ) DKA

a s in d ic a t e u , and a u to ra d io g r a p h e d . Tracks o f

a p a ir were n o t a u to ra d io g ra p h ed f o r eq u a l

d u r a t io n s . The tem p eratu re o f h y b r id is a t io n

was 4 5 ° .

RIGHT HAND SIX PAIRS OF TRACKS

A f f e c t o f tem p eratu re on the h y b r id is a t io n o f

n ic k t r a n s la t e d HSV-2 DNA to b lo t s t r i p s

c o n ta in in g Kpn I or liamH I d i g e s t s o f HSV-1

DNA.

jejI o t s t r i p s were h y b r id is e d w ith HSV-1 (1 ) or

HSV-2 (2 ) proue a t th ree d i f f e r e n t te m p e ra tu r es ,

th en autox’a u io g r a p h e d . Tracks o f a p a ir were n o t

a u to ra d io g ra p n ed fo r eq u a l d u r a t io n s .

Fragm ents o f tnu Kpn I d ig e s t are in d ic a te d on

tn e l e f t , ana damn I on the r i g h t .

IE CM

o COO 00L0

c CMa

IE CMCD mmmO

LA CQ<fr _

Ca CM

IE CMCD

o 00—ca CM

— I

-□■a "av-, E eo o a — . x — c

« • •

■ M S i i a ift P i » 4 l ■ ■ t" •

I *"§ CMm x o o) r'jj: — c a >.*< >x N<o T3 x E o o’ « > >

M R « n 1SOTJJl*. O) .* £X -*-■ _

co co

N CO O ) O * - <N CON W S C O O -J J ' J IA D*-c4 <0 —. 'rn'~ —0) *-

S p i n 0 'T t ® CO o> 5 2 « “ ? x £ ® ®Wirt *"’ Tf

Figure C2.5

Summary o f HSV-l/HSV-2 in t e r t y p i c hom ology d a ta .

A u to ra d io g ra p h s o f n i t r o c e l l u l o s e b lo t s t r i p s o f

HSV-1 or HSV-2 d i g e s t s to w hich n ic k t r a n s la t e d

HSV-1 o r HSV-2 DMA had been h y b r id is e d a t 45° were

a u t o d e n s i t o m e t r ic a l ly scanned and R^ v a lu e s c a lc u la t e d

( s e e t e x t ) . In t h i s diagram mean v a lu e s a re

r e p r e s e n te d h i s t o g r a p h ic a l ly w ith r e s p e c t to

r e s t r i c t i o n maps o f the- HSV genome in th e c o n v e n t io n a l

o r ie n t a t io n ( P ) . Fragm ents w hich c o u ld n o t be

sc o r e d a r e n o t in c lu d e d i n th e h is to g r a m . The

number o f a u to r a d io g ra p h s scanned f o r ea ch d i g e s t i s

shown on th e r ig h t o f each map, and th e v e r t i c a l l i n e

on th e l e f t in d ic a t e s an R^ v a lu e o f 1 . 0 . An

appx*oximate d i s t r i b u t i o n o f i n t e r t y p i c hom ology i s

g iv e n above th e genome a t th e bottom , where la r g e R

v a lu e s in d ic a t e r e g io n s o f h ig h e r hom ology; th e y a r e

c l a s s i f i e d in t o th r e e l e v e l s o n ly .

H i n d Hi

E c o R I

Bgl II

H p a I

Kp n I

P v u II

X h o I

B a m H I

HSV-2 B a m H I

Kp n I

H i n d / B g l

H i n d / E c o

H p a / X b a

Figure C2.4

A u to ra d io g ra p h s o f th e r e s u l t s o f h y b r id is a t io n

a t 4 0 ° o f n ic k t r a n s la t e d recom b in an t pAT153

p la sm id 3 c o n t a in in g i n s e r t s o f HSV-1 o r HSV-2 DNA

to b l o t s t r i p s o f HSV-2. BamH I ( l e f t ) o r HSV-1

BamH I ( r i g h t ) . The n a tu r e o f th e HSV DNA i n s e r t

i s in d ic a t e d a t th e top o f each, s t r i p .

HSV-

1 H

SV-2

HSV-

2 Hi

nd

III Ba

mH

I Ba

mH

I HS

V-2

Hind

III

CM

I I I

I I I

I t •

I I

roO<D-C '.Z' ~ r-C- -CO,™ J* C ro lo ots ’a) oilc —

Figure C2#5

C ross b lo t s o f HSV-1 Kpn I/HSV-

and HSY-1 BamH 1 / HSV-1 BamH I .

d im en sio n i s v e r t i c a l , and th e

d im en sion i s h o r iz o n ta l*

■1 BamH I (u pp er)

The u n la b e l le d

32p_iabelled

Kpn Ia

c fd 9hi j k Ii it 11 i i mop qrst u

BamH I

w y

. -

uv-

a -b -

c - d - e -

f 9 - h - i -

BamH I

BamH I

• Fig u r e C2»6

C ross b l o t s o f HSV-1 KcoK I/H SV -1 EooR I (u p p er)

and HSV-1 BamH l/H SV -2 BamH I ( lo w e r ) . The

u n la b e l le d d im en sio n i s v e r t i c a l , and th e ^ P - l a b e l l e d

d im en sio n i s h o r iz o n t a l .

EcoR I

EcoR I

c d e f 9

a h b ji i k Ii i mi

cdefghi

k—#I-*

m -

n -

HSV-2

f i] k I nI 411 opqri Mir

32 P HSV-1

*

Figure C2.7

Homology b etw een HSV and PRV o r EHV-1 UNA.

N ick t r a n s la t e d HSV-1 ( l ) , HSV-2 ( 2 ) , PRV (P) or

EHV-1 (E) DNA v/as h y b r id is e d a t two tem p era tu res

to b lo t s t r i p s o f HSV-1 BamH I o r HSV-2 BamH I ,

a s in d ic a t e d below th e p a n e ls . Exposure tim e

d i f f e r s betw een t r a c k s .

c

LU 1 $ I I i j

▼-

CM 0 #

# # i #

i f i |

c jp

o

§

*£ c a - 3X n <o« ^ r - p o c S 5 >

c >-O "O 03 *»-

ob» A '■— — 'c

-*

LU M i * t ■

CM •• • L t 1

p m K E 5 m •M B 0a o o S ^ ^ c o O’ »- V)~ s r v o t _* £ a > > X>N 3 > OTD03.O)IC —H-

LU i •Q . • • • • I I I 1 1

i si it m * iCM 0 * * *

oin

-Q 0 03 0 3 C O . 3 xw "O'4- A ~ OO" V) $ C >

03 03'o M- Oi'A '■-

E

LU II 9 t I0 - M l M ' • l •CM M i t • • iT— • 1 — m • • » 1

as 00303 •—> ~ CA -a -c ^ E

> 4 - —

O 0"i_ V)*->a uv w X V z '03 ~_Q "O T3 03030

HSV-

1 HS

V-2

HSV-

1 HS

V-2

Fig u r e C 2,8


a t 40° o f n ic k t r a n s la t e d recom b in an t pAT153

p la sm id s c o n t a in in g i n s e r t s o f HSV-1 o r HSV-2 DMA

to b lo t s t r i p s o f EHV-1 B g l >II/EcoH I ( l e f t ) or

B gl I I ( r ig h t ) d i g e s t s . The n a tu r e o f th e HSV

DiJA i n s e r t i s in d ic a t e d a t th e to p o f each s t r i p .

E xp erim en ts wei’e done t w ic e .

cc— + o o>T o CO LU

figure C2,9


a t 40° o f n ic k t r a n s la t e d recom b in ant pAT153

p la sm id s c o n t a in in g i n s e r t s o f HSV-1 or HSV-2 DNA

to b l o t s t r i p s o f PRV BamH I . ( l e f t ) or Kpn I

( r ig h t ) d i g e s t s . The n a tu r e o f th e HSV DNA

i n s e r t i s in d ic a t e d a t th e top o f each s t r i p .

E x p erim en ts were done t w ic e .

7 1 .......w i 05 rX CO

t- “ >-

MI ,2 -*GO<\l

OC

4 • • I I

l> • t tI • • •

0)CNI>C/)X

Ca*

<b.q o " o je —

1 1 in <o X co

SiCO

■oc

o> \ , • 1> •-* I • 1 • •5! t t • • •o ; •c ;

- m* • f• ••° A • • •

M 1 • • t * •__ l i ) UX

"O "JW-C - E e e

£•CO

F igure C2.10

Summary o f th e d i s t r i b u t i o n o f hom ologous r e g io n s

betw een th e genom es o f HSV-1, HSV-2, EHV-1 and PRV.

The upper p a n e l show s r e s t r i c t i o n maps o f th e HSV-1

and HSV-2 genom es i n th e 1^ a rran gem en t. B la ck r e c t a n g le s

in d ic a t e r e g io n s o f s i g n i f i c a n t l y g r e a t e r hom ology betw een

th e probe and th e b lo t s t r i p s to w h ich i t was h y b r id is e d

( p r o b e /b lo t ) , and h o r iz o n t a l l i n e s f o r EHV-1 and PRV p ro b es

show r e g io n s o f w eaker but d e t e c t a b le h y b r id i s a t io n .

R e s u lt s f o r HSV, EHV-1 and PRV p ro b es a re ta k en from

F ig u r e C 2 .3 , and T a b les C 2*2-C 2*3, u s in g h y b r id is a t io n

tem p er a tu re s o f 45° > 40° and 45° r e s p e c t i v e l y . The sev en

r e g io n s ( I - V I I ) o f g r e a te r hom ology betw een th e HSV-1 and

HSV-2 genom es a re in d ic a t e d , and th e c lo n e d HSV-2 H in d iI I

fra g m en ts w ith in w hich th e y a re lo c a t e d ( a , b , e , h , 1 2 ) .

The two lo w er pa n e l s show th e lo c a t io n s o f

r e s t r i c t i o n fra g m en ts w ith in th e PRV (P o w e ll , 1 9 7 9 ) and

EHV-1 genom es (J.M . W halley, p e r s o n a l com m unication) to

w hich c lo n e d HSV DNA fra g m en ts h y b r id is e d ( f o r n om en cla tu re

s e e T able B 1 . 3 ) . Data a re ta k en from T a b le s C 2 .4 -C 2 .5 ,

and fra g m en ts i n p a r e n th e se s d em o n stra ted w eaker h y b r id is a t io n .

Homologous r e g io n s i n th e EHV-1 genome a l ig n w ith

r e s p e c t to th o se in th e HSV genome i n th e 1^ (o r 13^)

arran gem en t, w h ereas th e PRV and IISV genom es a p p e a r 'n o t to

be c o l in e a r .

h s v -1 m i — — —------------------------------------------------ 1 n — c

BamH I I I I I I I I III 11 *1111 I I I I II I Mils b I c

PRV/HSV-1

EH V/HSV-1

HSV-VHSV-2

EHV/HSV-2-------

PRV/HSV-2 -------

I■a

■

II III IV V

m m f " be h

k y n i **vn

■ ■

■ ■

VII VII1 i

12 12

■

■ ■

m ^ k inv p t k w a t e m i y o m b I n s r i i d q h x c pgg i conaguBamH I I I I II I I I I 11 III II III I II II II l l l l Hill

HSV-2 C=J------------------------------------------ ----------------- [— 17)------- aHind III II I

i oII

h ni I I

i+nt I k

12,y.|k)a e h h,e b b 12,k,y,g

r - 1 »v 1 v v """i /' ■' "iBgl II I I I I I II II I I I I I I

k I d | I c o g x b h i n e m a12.g.(k)

a b.e.h b 12,k,y.gr-n /—---------------- —\/------------w a / >

EcoR I I I I I I II I I I I I Ij o g n m h p i a e l b k l

12.fl.lk). a e h h.e b b 1 2 (k,y,g

Bgl ll/EcoRI I I fi I Til I II !■* H V I I I I f i M ' I I ‘n I l b' I r m x o s y g / h d i q u v j p w a a(c) k e(b)

a e.h h 12.9jM b________ ^ 1 2 .k ,g—BamH I I I I M I I T l I I M I I I l l T

I <1 | h m I k a ■ b 1 c o e t | p d n r s

EHV

summary * M N p N M N * Ia e h (e) b b 12.k.y.g

12,g,(k)

BamH I

Kpn I

PRV

e.h

II* p

b ^ \ 1 2 . k . g ^ A

i i i 1 P ii i i 11 ii f ?b k m d c g q j i e i h n e i o

a h \n

h a.e/——~-\f — 1 ■I I

b ^ -^ .k .g .y -v11 ii i' 1i i i i r

k b g m f e I j i I h

---------------------------c

summary

d— m

MV

12.k ,g

Figure C 2 . l l

A u torad iograp h o f th e r e s u l t s o f h y b r id is a t io n

. a t 40° o f n ic k t r a n s la t e d HSV-2 ( 1 ,4 ) o r Cl.iV ( 2 ,3 )

BN A to n i t r o c e l l u l o s e b lo t s t r i p s c o n ta in in g th e

HSV-2 BamH I d i g e s t ( 1 ,2 ) or th e CMV Hind I I I

d i g e s t (3 > 4 ) . H e lev a n t r e s t r i c t i o n fra g m en ts

a re i n d ic a t e d , and r e s t r i c t i o n maps o f CMV

(AB169) BNA from W e s ts tr a te e t a l . (1 9 8 0 ) a re

shown, w ith a m o le c u la r w eig h t s c a le (x 1 0 ~^ d a l t o n s ) .

d imoqswy

c cef g

jkln

prtu

x z

a' bd*

e’

Figure C2«12

H istogram o f c r o s s o v e r p o s i t i o n s in th e L segm ent o f

reco m b in a n ts produced by marker r e s c u e o f HSV-1 tsD

w ith HSV-2 EcoRI j o in t fr a g m e n ts . 33 c r o s s o v e r s in

87 reco m b in a n ts o ccu rred a t th e l e f t hand s id e o f 1 ,

and 4 c r o s s o v e r s a t th e r ig h t hand s i d e .

HSV-2 r e s t r i c t i o n s i t e s a re shown a l ig n e d w ith r e s p e c t

to th o s e f o r HSV-1 below th e r e p r e s e n ta t io n o f th e

p o r t io n o f th e L segm ent co v ered by th e r e s c u in g

HSV-2 fr a g m e n ts . The 1 segm ent i s shown r e l a t i v e

t o th e P o r ie n t a t io n of- th e genom e. An e s t im a t io n

o f th e d e g r e e o f in t e r t y p i c hom ology i s shown by th e

t h ic k n e s s o f th e b la ck l i n e .

1 X bal

2 H indiX I

3 EcoR I

4 .B g l U

5 Hpal

6 Kpnl

7 BamHI

INTE

RTY

PIC

RECO

MBI

NA

NTS

of

HSV

-1 t

s D

syn*

/ HS

V-2

EcoR

I

join

t fr

agm

ents

;

CRO

SSO

VER

PO

INTS

IN

LONG

RE

GIO

N

oc

SECTION 3

NUCLEOTIDE SEQUENCES 01' THE JOINT

BETWEEN THE L AND S SEGMENTS

OP THE GENOMES OP HSV-1 AND HSV-2

- 107 -

RESULTS


The a seq u en ce i s p r e s e n t a s a d i r e c t r e p e t i t i o n a t

th e p h y s ic a l t e r m in i o f th e IISV genome, and a l s o in in v e r te d

o r ie n t a t io n a t th e j o in t between L and S . The o b j e c t iv e o f

t n i s work was to d eterm in e the DNA seq u en ce o f th e j o in t

r e g io n s o f th e genomes o f HSV-1 and HSV-2, in o rd er to

l o c a t e seq u en ce arran gem en ts w hich m igh t be in v o lv e d in th e

f u n c t i o n ( s ) o f th e a seq u e n c e , to e v a lu a te th e co d in g

p o t e n t i a l o f t h i s r e g io n , and to u n d ersta n d th e p r e v io u s ly

o b se rv ed s i z e h e t e r o g e n e i ty in th e HSV-1 j o i n t r e g io n

(Wagner and Summers, 1 9 7 8 ) .

S s t I r e s t r i c t i o n maps o f the HSV-1 and HSV-2 .jo in t r e g io n s

K eclea v a g e ex p er im en ts were perform ed to map th e

r e s t r i c t i o n s i t e s o f S s t I (or i t s is o s c h ia o m e r , S a cI )

w ith in th e llSV-1 and HSV-2 BamHI j o i n t and te r m in a l fr a g m e n ts .

I m p l i c i t in th e f o l lo w in g argum ents i s th e a ssu m p tio n th a t

r e s t r i c t i o n s i t e s in IR^ or IRg are p r e s e n t in i d e n t i c a l

l o c a t io n s in TR o r TRg', r e s p e c t iv e ly .

F ig u re C l ,1 8 shows th a t HSV-1 BamHI k c o n s i s t s o f a

f u s io n o f BamHI q and q , and th a t EcoRI k i s an S ter m in a l

fragm en t w hich e n t i r e l y c o n ta in s BamHI q . The s i z e s o f th e

fo u r S s t I fra g m en ts produced from HSV-1 BamHI k a re g iv e n in

T able C 3 * l, and no s m a lle r fragm en ts were d e te c te d on

p o ly a c r y la m id e g e l s . The r e s u l t s shown in F igu re C 3.1 p a n e l

A a llo w the p o s i t i o n s o f S s t I s i t e s w ith in EcoRI k and BamHI k

to be d e term in ed . Bamll l s gave two fr a g m e n ts , th e la r g e r

o f w hich co m ig ra ted w ith S s t I B ( tr a c k 2 ) . T h erefore th e

s in g l e S s t I s i t e w ith in BamHI q d e f in e s S s t I B a t th e l e f t

hand end o f BamHI k , w ith r e s p e c t to th e c o n v e n t io n a l

4

- 108 -

arrangem ent o f th e genom e, EcoRI k produced th r e e fra g m en ts ,

th e s m a l le s t o f w hich co m ig ra ted w ith S s t I C ( tr a c k 3 ) ,

Only th e la r g e r o f th e o th e r two fr a g m en ts co m ig ra ted w ith

a fra g m en t in th e HSV-1 S s t I d i g e s t (n o t show n), and so

t h i s i s th e S te r m in a l S s t I fra g m en t. T h ere fo re S s t I A

m ust be th e j o in t fra g m en t, c o m p r is in g a f u s io n o f th e

s m a lle r S s t I fragm en t o f BamHI s p lu s th e l a r g e s t S s t I

fragm en t o f EcoRI k . To th e r ig h t o f S s t I A i s C, w hich

wa3 common to th e BamHI k and EcoRI k d i g e s t s . To th e r ig h t

o f S s t I C i s B, th e r ig h t hand end fragm en t o f BamHI k .

T h ere fo re th e o rd er o f S s t I r e s t r i c t i o n fra g m en ts w ith in

HSV-1 BamHI k i s B A C B. F ig u re C 3.3 show s th e S s t I

p h y s ic a l map o f HSV-1 BamHI k .

HSV-2 BamHI g i s th e j o in t fragm en t c o n s i s t in g o f a

f u s io n o f BamHI u and v , EcoRI k and m a r e th e two S te r m in a l

fr a g m e n ts , and EcoRI f i s an 1 te r m in a l fr a g m e n t. T h erefore

BamHI u i s c o n ta in e d e n t i r e l y w ith in EcoRI k and m, and

BamHI v w ith in EcoRI f (F ig u r e C 1 .1 9 ) . BamHI g produced

e ig h t S s t I fr a g m e n ts , o f w h ich £ and 8 w ere more r e a d i ly

r e s o lv e d on p o ly a c r y la m id e g e l s ; fragm en t s i z e s a r e g iv e n in

T able C 3 .1 . F ig u re C 3.1 shows d a ta f o r th e d e d u c tio n o f

S s t I s i t e s w ith in BamHI g., BamHI u produced S s t I 1 , Jj, 6 ,

and, ta k in g in t o a cc o u n t band i n t e n s i t i e s , a fragm en t w hich

c o m ig r a te d w ith £ (F ig u r e C 3.1 p a n e l B tr a c k 2 ) . The

l a t t e r fragm en t m ust be th e S te r m in a l S s t I fra g m en t.

S s t I 6 was n o t produced by EcoRI k o r m and t h e r e fo r e m ust be

th e r ig h t hand fragm en t o f BamHI g , w ith r e s p e c t to th e

c o n v e n t io n a l r e p r e s e n ta t io n o f th e genom e. C lea v a g e o f

HSV-2 Kpnl a , w hich c o n ta in s th e e n t ir e S segm ent e x c e p t fo r

th e te r m in a l 1 x 1 0 6 d a lto n s ( i . e . Kpnl r , s e e F ig u re C l .1 9 ) ,

gave S s t I £ (F ig u re C 3.1 p a n e l D ), w hereas Kpnl r s g a v e ,

- 109 -

among o th e r p r o d u c ts , S s t I 8 (p a n e l E ) . T h ere fo re th e

o rd er o f S s t I fra g m en ts a t th e r ig h t hand s id e o f BamHI g

i s 8 1 £ 6 . S s t I £ a*1*1 1 o r ig in a t e , th e n , from th e l e f t

hand s id e o f BamHI g , and 2 was n o t produced by r e c le a v a g e

o f la r g e r te r m in a l fr a g m e n ts , s u g g e s t in g t h a t i t i s th e S s t I

j o in t fr a g m e n t. EcoRI f gave S s t I 2. an(1 Z n o t £> 311(1

t h e r e fo r e £ maps a t th e l e f t hand end o f BamHI g . In

c o n c lu s io n , th e o rd er o f S s t I fra g m en ts w ith in BamHI g i s

£ (2>Z) 2 8 1 ^ 6 , w ith 2 mapping a t th e j o i n t . The r e l a t i v e

o rd er o f 2 and Z was a s s ig n e d i n th e e lu c id a t io n o f th e

Smal map, and th e f u l l S s t I map i s shown in F ig u re C 3 .3 .

O ther r e s t r i c t i o n mans o f th e HSV-1 and HSV-2 j o in t r e g io n s

O ther maps w ere d eterm in ed l a r g e l y u s in g BamHI j o in t

fra g m en ts i s o l a t e d from recom b in ant p la s m id s . The major

form o f j o in t p r e s e n t in HSV-1 and HSV-2 DNA c o n ta in s a

s in g l e a se q u e n c e , but m inor form s c o n ta in two or more tandem

r e p e a te d a s e q u e n c e s . T h is d i s t r i b u t i o n was r e f l e c t e d in

c lo n e d fr a g m e n ts . The two p la sm id s u sed f o r d ed u c tio n o f

r e s t r i c t i o n maps were k l (HSV-1) and g l (HSV-2) (F ig u r e C 3 .6 ) .

The form er h a s a s in g l e a seq u en ce and th e l a t t e r has tw o .

F u rth er e x p e r im e n ts , u s in g a p la sm id w ith two a seq u en ces

i n th e HSV-1 DNA i n s e r t (1E2 in F ig u re C3#6) and a p lasm id

w ith a s in g l e a seq u en ce i n th e HSV-2 DNA i n s e r t , con firm ed

t h a t m u lt ip le a se q u e n c e s a re p r e s e n t i n each c a s e a s tandem

d i r e c t r e p e a t s . T h is i s d is c u s s e d e ls e w h e r e . R e s t r ic t io n

maps shown in F ig u r e C 3.3 show s in g le a se q u e n c e s a t th e j o i n t .

P la sm id DNA was n ic k t r a n s la t e d , c le a v e d w ith BamHI ,

and th e r e le v a n t j o i n t fragm en t i s o l a t e d from an a g a ro se g e l .

A number o f s in g le and d ou b le d ig e s t io n s were th en done to

map r e s t r i c t i o n s i t e s , a s w e l l a s some p a r t i a l d ig e s t io n s

and d ig e s t io n s o f 5 1- t e r m in a l ly l a b e l l e d DNA. P rod u ct s i z e s

- 11 u -

were c a l ib r a t e d u s in g th e H in fI and H a e l l l d i g e s t s o f

pBK322 DNA ( S u t c l i f f e , 1 9 7 8 ) . The d a ta i s shown in T a b les

C 3.2 and 0 3 -3 , from w hich r e s t r i c t i o n maps were deduced

(F ig u r e C 3 .3 ) . D e t a i l s o f th e argum ent f o r th e Smal map

o f HSV-1 BamHI k a re d e s c r ib e d b elow .

The H in c II and Sau3AI s i t e s in BamHI k were lo c a t e d

(T a b le C 3*2). P a n e ls A and B o f F ig u re 03*2 show d ou b le

d ig e s t io n s o f HSV-1 BamHI k in c lu d in g Sm al. H inc I I c le a v e d

Smal B, S s t I c le a v e d Smal B, E and N, and Sau3AI c le a v e d

Smal A and B. C lea v a g e o f i s o l a t e d S s t I and Sau3AI

fra g m en ts a llo w e d Smal fra g m en ts to be a s s ig n e d to r e g io n s

o f BamHI k a s f o l lo w s (T ab le C 3 ,2 ) .

i f W fi A (F ,R ) B (Mf fl) E 0

32The o rd er w ith in Sau3AI A was d eterm in ed by P - l a b e l l i n g

t h i s fragm en t a t th e 5 1 te r m in i w ith T4 p o ly n u c le o t id e k in a s e ,

d ig e s t in g w ith S s t I and s e p a r a t in g th e two u n iq u e ly la b e l l e d

fra g m en ts by a g a ro se g e l e l e c t r o p h o r e s i s . The two fra g m en ts

were th en p a r t i a l l y d ig e s t e d w ith Smal and s i z e s o f p ro d u c ts

d eterm in ed by com fjarison w ith m o le c u la r w e ig h t m arkers

(F ig u r e C 3.2 p a n e l F ) , The p ro d u ct s i z e s co rresp o n d to

s e q u e n t ia l a d d it io n s o f Sima I fra g m en ts to th e l a b e l l e d

te r m in a l Smal fra g m en t, so th a t th e s i z e in c r e m e n ts co rresp o n d

to Smal fragm en t s i z e s . The p a r t i a l d ig e s t io n p ro d u c ts a re

shown in T able C 3 .2 , and g iv e th e o rd er o f Smal fra g m en ts

w ith in Sau3AI A a s J C L D N H I K P G , w ith 430 bp o f

Smal A betw een Smal G and th e Sau3AI s i t e ( th e Sau3AI s i t e

c o in c id e s w ith th e BamHI s i t e a t th e l e f t s id e o f J ) .

The o n ly a m b ig u ity i s in th e p o s i t i o n s o f Smal G and H,

s in c e th ey a re s im i la r i n s i z e . They were unam biguously

a s s ig n e d by f i n e mapping o f th e H in f l fragm en t c o n ta in in g

- I l l -

Smal A, G, K, P and p a r t o f I , w hich i s d e s c r ib e d l a t e r .

The o n ly fragm en t common to th e j o i n t and b oth te r m in i was

Smal G (T ab le C 3 .2 ) , s u g g e s t in g th a t t h i s fragm en t i s

c o n ta in e d c o m p le te ly w ith in th e q se q u e n c e .

The o rd er o f Smal F and R was a s c e r ta in e d by p a r t i a l l y

d ig e s t in g Sau3AI B (F ig u re C 3.2 p a n e l C ), The o rd er o f

fra g m en ts w ith in Sau3AI B from d ou b le d ig e s t io n i s , in bp,

6 4 0 - (2 4 ,4 5 0 ) - 4 9 5 , where th e unordered fra g m en ts Smal F and R

a re 450 and 24 bp r e s p e c t iv e ly (a s c a l ib r a t e d on a g a r o se ;

F m ig ra ted a s 410 bp on p o ly a c r y la m id e ) . The

p r e d ic te d p a r t i a l d ig e s t io n p r o d u c ts from th e two p o s s ib l e

arran gem en ts a re shown in T ab le C3*2 p a r t I . The th r e e

la r g e r p a r t i a l d ig e s t io n p ro d u c ts were 1 1 1 5 , 970 and 945 bp

(F ig u r e C 3.2 p a n e l C ), .su p p o rtin g th e o r d e r o f th e two

fra g m en ts a s Smal R F. The o th e r two e x p e c te d p a r t i a l

d ig e s t io n p ro d u cts o f 665 and 475 bp were n o t d i s t in g u is h e d

p ro b a b ly b ecau se 665 bp d id n o t r e s o lv e from 640 bp, and

475 bp d id n o t r e s o lv e from 495 bp. The i n t e n s i t i e s o f th e

bands a t 640 and 495 bp were n o t d e c r e a se d to th e same

e x t e n t a s th e 450 bp (Sm al F) band by u s in g l e s s Smal in th e

d i g e s t i o n . The o rd er was con firm ed a s R F by p a r t ia l

d ig e s t io n o f S s t I A, when F-R was d e t e c te d b ut n o t 750-R ,

where 750 bp i s th e p a r t o f Smal B c o n ta in e d w ith in S s t I A

(F ig u r e 0 3 ,2 p a n e l E ) .

Smal M and £ were o rd ered by p a r t i a l d ig e s t io n o f S s t I C.

The o rd er o f fra g m en ts w ith in S s t I C from d o u b le d ig e s t io n

i s , in bp, 7 8 - ( 9 1 ,3 8 ) - 2 6 0 , where th e u n ord ered fra g m en ts

Smal M and a re 91 and 38 bp, r e s p e c t i v e l y . The p a r t i a l

d ig e s t io n p r o d u c ts p r e d ic te d from th e two p o s s ib l e arran gem en ts

- 112 -t

a r e shown in T ab le C 3.2 p a r t J . The a c t u a l p r o d u c ts , shown

in F ig u re C 3.2 p a n e l D, g iv e th e o rd er a s Smal M The f u l l

o rd er o f Smal fra g m en ts in HSV-1 BamHI k i s shown in F ig u re

C 3 .3 . The o th e r r e s t r i c t i o n maps shown in t h i s F ig u re were

d eterm in ed s im i l a r l y by c o n s id e r a t io n o f th e d a ta in T a b le s

C 3.2 and C 3 .3 .

Si z e v a r i a b i l i t y in th e .jo in t r e g io n

R e c e n t ly p la q u e -p u r if ie d s to c k s o f HSV-1 s t r a in 17 and

HSV-2 s t r a in HG52 were p la te d on BHK c e l l s , grown under

Biiu2, and a s in g le p laq u e o f each p ic k e d . Ten su b p la q u es

o f each were i s o l a t e d , p la q u e -p u r if ie d t w ic e m ore, and a

v ir u s s to c k p rep ared a t 37° £ o r each p a r e n t and progeny■50

p la q u e . V ir io n DMA was P - la b e l l e d in v i v o and BamHI

r e s t r i c t i o n p r o f i l e s compared (F ig u r e C 3 .4 ) . T erm inal

and j o i n t fra g m en ts o f HSV-1 DNA w ere th e m ost v a r ia b le in

s i z e (BamHI k , q , s ) , and v a r ia t io n s i n th e j o i n t and S

te r m in a l fra g m en ts (BamHI k and q , r e s p e c t i v e l y ) seemed

c o r r e la t e d . V a r ia t io n was shown to be in q and n o t p ,

w hich co m ig ra ted i n some c a s e s , by d ig e s t io n w ith o th e r

r e s t r i c t i o n e n d o n u c le a se s . L ess o b v io u s v a r ia t io n was

ob serv ed i n th e m o b i l i t i e s o f HSV-1 BamHI n and x , w hich

span th e ju n c t io n s betw een Ug and TRg/IRg ( e . g . compare

tr a c k s 2 and 5)., .and in BamHI z , w h ich maps i n Ug ( e . g .

compare tr a c k s 5 and 6 ) . In c o n t r a s t , HSV-2 i s o l a t e s

v a r ie d o n ly in BamHI z and a 1, w hich span th e ju n c t io n s

betw een U3 and TR^/IR^, and no v a r ia t io n i n HSV-2 j o in t

(BamHI g) o r te r m in a l fra g m en ts (BamHI u and v ) was

d e t e c t e d . V a r ia b i l i t y was shown to r e s id e in z and n o t

y by d ig e s t io n w ith o th e r r e s t r i c t i o n e n d o n u c le a se s .

The r e g io n o f v a r i a b i l i t y in th e HSV-1 j o in t r e g io n

was lo c a t e d more p r e c i s e l y by Smal d ig e s t io n o f BamHI

- 113 --• . 32j o i n t fra g m en ts i s o l a t e d from in v iv o P—l a b e l l e d v ir io n .

Dll A (P ig u re C 3 .5 )« The j o in t r e g io n o f c l o n a l l y r e la t e d

HSV-2 i s o l a t e s was a n a ly se d s im i la r ly (P ig u r e C 3 .6 ) .

I1SV—1 Smal G v a r ie d in s i z e betw een 275 and 400 bp see m in g ly

by th e in s e r t i o n or d e l e t io n o f m u lt ip le s o f a p p ro x im a te ly

1 1 -1 2 bp. HSV-1 Smal A e x h ib ite d s i z e v a r i a b i l i t y o f up to

400 bp w hich was in d ep en d en t o f th a t in Smal G. D ig e s t io n

o f BamHI k from f i v e o f th e HSV-1 i s o l a t e s w ith Sau3AI and

Tag I a llo w e d th e v a r ia b le r e g io n i n Smal A to be more

a c c u r a t e ly lo c a t e d betw een th e Sau3AI A-B and Tag I A-D s i t e s

(P ig u r e 0 3 * 5 , maps in P ig u re C3*3)* R e c lea v a g e o f BamHI

fra g m en ts c o n ta in in g th e genom ic t e r m in i show ed, f o r each

i s o l a t e , th e same s i z e d i s t r i b u t i o n o f v a r ia b le fra g m en ts

a s in th e j o in t r e g io n . No v a r ia t io n i n th e BamHI j o i n t

fra g m en ts o f th e HSV-2 i s o l a t e s was d e t e c te d by c le a v a g e

w ith Smal (P ig u re 0 3 .6 ) or S s t I .

HSV-1 BamHI j o i n t fra g m en ts i s o l a t e d from p la sm id s ,

l i k e th o se i s o l a t e d d i r e c t l y from v ir io n DNA, showed s i z e

v a r ia t io n in Smal A and G (P ig u r e C 3 .6 ) , w hereas no v a r ia t io n

was o b serv ed in c lo n e d HSV-2 j o i n t fr a g m e n ts , e x c e p t th a t

due to th e p r e se n c e o f an e x tr a a seq u en ce a s d e s c r ib e d

b e lo w . One o f th e HSV-1 c lo n e s , k 2 , had a s m a lle r Smal I

fra g m en t, a lth o u g h v a r i a b i l i t y in t h i s fragm en t was n o t

o b serv ed in v ir io n DNA.

A lthough HSV-1 and HSV-2 d i f f e r in th a t th e form er

showed s i z e v a r i a b i l i t y in th e j o i n t r e g io n and th e l a t t e r

d id n o t , th e y both p o s s e s s a secon d ty p e o f h e t e r o g e n e ity

in th e j o in t r e g io n and a t th e L term in u s w hich c o n s i s t s o f

i n s e r t i o n s o f a p p r o x im a te ly 300 bp th o u g h t to c o n ta in th e

a s e q u e n c e . The m a jo r ity o f HSV DNA m o le c u le s h as a s in g l e

a seq u en ce a t th e j o i n t , w h ile a s m a lle r p r o p o r t io n h a s two

- 114 -

or m ore. T h is i s r e f l e c t e d in c lo n ed HSV-1 j o in t fra g m en ts ,

m ost o f w hich had a s in g le a seq uence (kl, k 2 , k3 in P igu re

C 3 .6 ) , but some o f w hich had two (El, 3E2). P la sm id s El and

§2 each had two Smal G fra g m en ts , th e l a t t e r w ith two o f th e

same s i z e but th e form er w ith two o f d i f f e r e n t s i z e s . Smal

p r o f i l e s were c o n s is t e n t w ith the p r e se n c e o f th e two a

se q u e n c e s a s tandem d i r e c t r e p e a ts ( s e e P ig u re C 3 .1 3 ) .

S im ila r r e s u l t s were o b ta in ed w ith c lo n e d HSV-2 j o in t fr a g m e n ts ,

and an i s o l a t e w ith two a seq u en ces ( g l ) i s in c lu d e d in

P ig u re C3*6.

Homology betw een th e .jo in t r e g io n s o f HSV-1 and HSV-2 DNA

N it r o c e l lu lo s e b lo t s c o n ta in in g Smal fra g m en ts o f

HSV-1 and HSV-2 BamHI j o in t fragm en ts were p rep ared from

p la sm id DNA (k l and g l .in P igu re C 3 .6 ) by t r a n s f e r from 2 f*

a g a ro se or 5# p o ly a cry la m id e g e ls * S t r ip s were probed w ith

n ic k t r a n s la t e d p lasm id DNA c o n ta in in g th e j o i n t fragm en ts*

R e s u lt s from p o ly a cry la m id e g e l s a re shown in P ig u re C 3 .7 .

fra g m en ts la r g e r than 1000 bp and s m a lle r th an 150 bp d id n o t

t r a n s f e r e f f i c i e n t l y , and th e r e fo r e o n ly HSV-1 Smal B -J and

HSV-2 Smal 2 -6 were r e s o lv e d a f t e r h y b r id is a t io n . The

l a r g e r fra g m en ts t r a n s fe r r e d s a t i s f a c t o r i l y from a g a ro se

g e l s . Homologous p rob es f a i l e d to show e x p e c te d h y b r id is a t io n

to fra g m en ts mapping in th e a seq u e n ces (HSV-1 Smal G, HSV-2

Smal £ and J) and so i t was n o t p o s s ib le to compare hom ology

betw een th e iiSV-1 and HSV-2 a s e q u e n c e s . T h is e f f e c t co u ld

have been due e i t h e r to i n e f f i c i e n t t r a n s f e r o f th e s e

fra g m en ts to n i t r o c e l l u l o s e or to i n e f f e c i e n t h y b r id is a t io n .

HSV-1 p lasm id probe h y b r id ise d to HSV-2 Smal 1 (a g a r o se

g e l s ) and £ , and HSV-2 probe h y b r id is e d to HSV-1 Smal

B, S, P and H (th e S s t I/S m al double d i g e s t showed th a t

h y b r id is a t io n d id n o t occur to D ). These r e s u l t s are

- 115 -

in te r p r e te d , in term s o f a r e g io n o f i n t e r t y p i c hom ology

e x te n d in g th ro u g h HSV-1 Smal P, B and B (HSV-2 Smal 1 ) in

th e c s e q u e n c e s , and a r e g io n o f l e s s hom ology betw een HSV-1

Smal H and HSV-2 Smal ± in th e b s e q u e n c e s .

P in e r e s t r i c t i o n maps o f th e .jo in t r e g io n s o f HSV-1 and HSV-2

B e fo r e a t te m p tin g to d eterm in e th e n u c le o t id e seq u e n ces

o f th e j o in t r e g io n s o f HSV-1 and HSV-2 DNA i t was n e c e s sa r y

to map f u r t h e r r e s t r i c t i o n s i t e s i n t h e s e r e g io n s . N ick

t r a n s la t e d HSV-1 BamHI k Sau5AI A from p la sm id k l was

c le a v e d w ith a number o f e n d o n u c le a se s and a l s o d ou b le

d ig e s t e d w ith Smal . H in fl a p p a r e n t ly d id n o t c le a v e Smal

G, K, P and 430 bp (p a r t o f Smal A) b ut d id c le a v e Smal I

(P ig u r e C 3.8 p a n e l A ). I s o l a t io n o f th e l a r g e s t Sau3 A l/H in fI

fragm en t o f 1130 bp and* d ig e s t io n w ith Smal showed t h a t i t

c o n ta in e d Smal G, K, P and two fr a g m en ts o f 410 bp (430 bp

on a g a r o se ; p a r t o f Smal A) and 160 bp (p a r t o f Smal I ) .

The l a r g e s t Hinf l / BamHI fragm en t from p la sm id k l (a H in fl

fragm en t o f 1760 bp) c o n ta in e d Smal G, K, P and two fra g m en ts

o f 1040 bp (n e a r ly a l l o f Smal A) and 160 bp (p a r t o f Smal I ) .

A number o f s in g le and d o u b le d i g e s t s were done to map t h i s

fra g m en t, w hich c o n ta in s th e HSV-1 j o i n t a se q u e n c e . D ata a re

summarised in Table 0 3 .4 , and r e s t r i c t i o n maps a re

shown i n P ig u re C 3 .9 . HSV-2 BamHI g S s t I 2 and Smal 2 were

mapped s im i la r ly ( s e e P ig u r e 03*3 f o r th e l o c a t io n s o f t h e s e

fr a g m e n ts ) . D ata a re summarised i n T ab le 0 3 . and maps

a r e shown in P ig u re 0 3 .9 .

Lo c a t io n o f th e v a r ia b le r e g io n w ith in th e HSV-1 a seq u en ce

P ig u re 0 3 .1 0 shows a com parison o f d i g e s t s o f th e

Sau3 A l/Hinf l j o in t fragm en t from p la sm id s k l and k 2 , in th e

- 116 -

l a t t e r o f w hich Smal G i s la r g e r (P ig u r e C 3 .6 ) . The fra g m en ts

w hich a re la r g e r in k2 than in k l a r e , in hp, Smal - 340 ,

A v a il - 680 , B stN I - 1 0 0 0 , A val - 3 4 0 , Hael l - 9 8 0 , B g l l - 9 3 0 ,

S s t l l - 320 ( k l s i z e s g iv e n ) . The s i z e d i f f e r e n c e i s ab ou t

40 bp. The S s t l l r e s u l t p la c e s th e r e g io n o f v a r i a b i l i t y

w ith in th e a seq u en ce in th e 320 bp fr a g m e n t. Taken in

c o n ju n c t io n w ith th e Smal r e s u l t , th e v a r ia b le r e g io n r e s id e s

i n th e 270 bp betw een th e Smal s i t e a b o u t 40 bp from th e

l e f t hand end o f th e a seq u en ce and th e S s t l l s i t e ab ou t

100 bp from i t s r ig h t hand end , a c c o r d in g to th e maps shown

in P ig u re C 3 .9 .

De f i n i t i o n o f th e genom ic te r m in i and lo c a t io n o f th e a seq u en ce

P ig u re 0 3 .9 show s th e s t r a t e g i e s a d op ted to d eterm in e

th e n u c le o t id e se q u e n c e s shown i n P ig u re 0 3 * 1 1 . S eq u en ces

were d eterm in ed f o r th e j o i n t r e g io n s o f HSV-1 s t r a i n s 17

and USA-8 and HSV-2 s t r a in HG52, and a l s o f o r th e r e g io n

sp an n in g th e ju n c t io n betw een two d i r e c t l y r e p e a te d a se q u e n c es

in HSV-1 s t r a in 17 and HSV-2 s t r a in HG52.

The a seq u en ce i s d e f in e d a s th e seq u en ce p r e s e n t a s

d i r e c t r e p e a t s a t th e genom ic te r m in i and a s an in v e r te d

r e p e a t a t th e th e j o i n t betw een th e 1 and S seg m en ts . Hence

th e te r m in i may be a l ig n e d w ith th e j o in t r e g io n a s shown in

P ig u re 0 3 .1 3 . The p o s i t i o n o f th e a seq u en ce a t th e j o i n t

ca n n o t be d eterm in ed d i r e c t l y from th e j o i n t n u c le o t id e

se q u e n c e , but i t can be d eterm in ed by l o c a t in g th e t e r m in i

w ith r e s p e c t to th e j o in t n u c le o t id e se q u e n c e , u s in g t h e i r

common r e s t r i c t i o n s i t e s . I t i s assum ed, a lth o u g h i t h as

n o t been p ro v en , t h a t th e te r m in a l and j o i n t a se q u e n c e s are

i d e n t i c a l .

ilSV-1 DIM A was ^ P -^ L ab elled s p e c i f i c a l l y a t th e 5 1

- 117 -

te r m in i and s i z e s o f genom ic te r m in a l r e s t r i c t i o n fra g m en ts

d eterm in ed (P ig u r e C 3 .1 2 ) . T h is method l o c a t e s th e 5 *

te r m in i o f th e genome, and n o t th e 3 ' t e r m in i , w ith r e s p e c t

to th e j o i n t s e q u e n c e . The s i z e s in n u c le o t id e s f o r th e

S term in u s o f s t r a in 17 w ere: Smal 3 6 -3 7 , H a e l l l 2 3 -2 4 .

The d i f f e r e n c e in s i z e o f t h e s e two fra g m en ts i s two

n u c le o t id e s l e s s th an would be p r e d ic te d from th e j o in t

n u c le o t id e seq u en ce in P ig u re 0 3 * 1 1 , in w hich th e Smal and

H a e l l l s i t e s are lo c a t e d a f t e r n u c le o t id e s 367 and 352

r e s p e c t i v e l y . T h is d isc r e p a n c y i s p ro b a b ly a r e s u l t o f a

h ig h p r o p o r t io n o f C m o ie t ie s (1 0 /1 5 ) in th e l a b e l l e d s tra n d

betw een th e two s i t e s , w hich in c r e a s e s th e o v e r a l l ch arge to

mass r a t i o o f th e la r g e r fra g m en t, c a u s in g i t to m ig ra te

f a s t e r in th e g e l . I f t h i s i s s o , th e s m a lle r Hael l l

te r m in a l fragm en t p r o v id e s th e more a c c u r a te e s t im a t e ,

p la c in g th e S term in u s a t 3 2 9 -3 3 0 on th e j o in t se q u e n c e .

The s i z e s in n u c le o t id e s f o r th e L term in u s i n s t r a in 17

w ere: S s t l l 1 5 -1 6 , Hael l l 9 -1 0 , Smal 6 -7* T hese r e s t r i c t i o n

s i t e s a re lo c a t e d in th e j o i n t seq u en ce a f t e r n u c le o t id e s

7 1 1 , 719 and 722 r e s p e c t i v e l y , b e a r in g in mind t h a t in

t h i s c a s e th e l a b e l l e d s tr a n d i s com plem entary to t h a t

shown in P ig u re C 5 . l l . Por th e r e a so n d e s c r ib e d above th e

sm a lle r fra g m en ts m igh t p r o v id e a b e t t e r e s t im a te o f th e

n u c le o t id e s d e f in in g th e 1 term in u s (7 2 8 -7 2 9 ) . The HSV-1

a seq u en ce in p la sm id k l t h e r e fo r e has a s i z e o f 3 9 8 -4 0 0 bp

(betw een 3 2 9 -3 3 0 and 7 2 8 -7 2 9 ) .

The genom ic te r m in i o f HSV-1 s t r a i n USA- 8 were d e f in e d

by th e same c r i t e r i a (P ig u r e C 3 .1 2 ) and a r e shown in P ig u re

C3 . l l a t 3 3 0 -3 3 1 and 7 2 8 -7 2 9 w ith r e s p e c t to th e s t r a i n 17

se q u e n c e . Prom th e a u to ra d io g r a p h rep rod u ced in P ig u re

- 118 -03*12 i t co u ld o© se e n t h a t S te r m in a l fra g m en ts from s t r a in

USA—8 D1VJA were two n u c le o t id e s s m a lle r th an th o s e from s t r a in

17 DNA, w hereas B te r m in a l fra g m en ts were th e same s i z e i n

b o th s t r a i n s . The S and L te r m in i o f HSV-2 s t r a in HG52

were lo c a t e d a t 6 2 -6 4 and 309 -3 1 9 r e s p e c t i v e l y (P ig u r e C 3 .H )

by Smal c le a v a g e o f n ic k t r a n s la t e d HSV-2 BamHI u and v

i s o l a t e d from v ir io n DNA (T a b le C3*3 p a r t s B and C ). The

HSV-2 a seq u en ce th e r e fo r e has a s i z e o f 2 4 5 -2 5 7 bp.

In b oth HSV-1 s t r a in s and th e HSV-2 s t r a in th e genom ic

te r m in i a re lo c a te d c l o s e to th e r ig h t hand end o f d ir e c t

r e p e a t s in th e j o in t s e q u e n c e s . The d i r e c t r e p e a t in s tr a in -

USA- 8 c o m p r ise s th e f i r s t 17 n u c le o t id e s o f th e 21 n u c le o t id e

d i r e c t r e p e a t in s t r a i n 17 a t 3 1 1 -3 3 1 and 710- 7 3 0 .

However, th e prim ary seq u e n ces o f th e HSV-1 d i r e c t r e p e a t s

a re d i f f e r e n t from t h a t o f HSV-2.

The n u c le o t id e seq u en ce o f p la sm id s c o n ta in in g j o i n t

fra g m en ts o f HSV-1 3t r a in 17 o r HSV-2 s t r a i n HG52 w ith two

a se q u e n c es showed th a t th e a se q u e n c es a re tandem d i r e c t

r e p e a t s w ith a s in g l e copy o f th e a p p r o p r ia te 21 o r 17

n u c le o t id e d i r e c t r e p e a t a t th e a - a ju n c t io n (P ig u r e 03*15)*

Tandem r e i t e r a t i o n s in th e j o i n t r e g io n

The HSV-1 s t r a in 17* seq u en ce shown in P ig u re Q 3*H

was d eterm in ed u s in g p lasm id k l , but k2 was a l s o seq uenced

in th e r e g io n s where i t d i f f e r e d in s i z e from k l . Three

r e g io n s o f r e i t e r a t i o n were found in th e HSV-1 s t r a in 17

j o i n t r e g io n , one lo c a t e d in th e a seq u en ce and two in

a d ja c e n t c seq u e n c es ( r e i t e r a t i o n s I , I I and I I I i n P ig u r e s

C 3.9 and C 3 .H ) . The th r e e r e p e a te d u n i t s o f 1 2 , 16 and 17

n u c le o t id e s o f w hich th e r e i t e r a t i o n s c o n s i s t have d i f f e r e n t

prim ary s e q u e n c e s , a lth o u g h th e y a l l c o n ta in a predom inance

- 119 -

o f C r e s id u e s in th e same s tr a n d o f th e d u p le x . The seq u en ce

o f k2 in two o f th e r e i t e r a t i o n s ( I and I I I ) d i f f e r e d from

t h a t o f k l o n ly in th e number o f r e i t e r a t e d u n i t s .

R e i t e r a t io n I (4 0 8 -6 2 3 ) iu k l c o n ta in e d 18 c o p ie s o f

CCGCTCCTCCCC w hereas k2 c o n ta in e d 21 c o p ie s (P ig u r e C 3 .1 4 ) .

S im i la r ly , r e i t e r a t i o n I I I (1 1 6 7 -1 4 8 9 ) c o n s is t e d o f 19

c o p ie s o f CCGCCCCTCGCCCCCTC in k l and 11 c o p ie s in k2

(P ig u r e 0 3 * 1 5 ) . T hese two r e i t e r a t i o n s a re c o n ta in e d

w ith in Smal G and Smal A r e s p e c t i v e l y , and p r o v id e an

e x p la n a t io n fo r th e o b serv ed s i z e v a r i a b i l i t y in th e a and

c seq u e n c e s o f d i f f e r e n t p la q u e i s o l a t e s o f HSV-1 s t r a in 1 7 .

R e i t e r a t io n I I (9 2 8 -1 0 5 5 ) d id n o t c o n ta in a s im p le

r e p e a te d u n i t . F u rth erm ore, th e d i f f e r e n c e in seq u en ce

betw een k l and k2 was n o t r e f l e c t e d in d i f f e r e n t copy

numbers but in th e o r g a n is a t io n o f th e r e p e a te d u n i t s , th u s:

k l (CCCTCCCCAGCCCCAG)2 (CCCTCCCCGGCCCCAGJs

k2 (CCCTCCCCAGCOGCAG)! (CCCTCCCCGGCCCCAGjy.

The two 16 n u c le o t id e r e p e a te d u n i t s d i f f e r in a s in g l e

n u c le o t id e , and th e seq u e n c es o f r e i t e r a t i o n I I in k l and k2

d i f f e r by a s in g l e n u c le o t id e (P ig u r e C 3 .1 4 ) .

R e i t e r a t io n s I and *11 were a l s o i d e n t i f i e d in , th e

j o in t r e g io n o f liSV-1 s t r a in USA-8. R e i t e r a t io n I I

com p rised a t l e a s t 15 c o p ie s o f th e f i r s t 16 n u c le o t id e

v a r ia n t o f th e r e p e a te d u n i t o f s t r a in 17 (shown a b o v e ) .

R e it e r a t io n I i s r e la t e d to t h a t o f s t r a in 17 in a more

com plex way (P ig u r e 0 3 .1 4 ) . P ig u re C 3 . l l shows t h a t i t

c o n ta in s 8 c o p ie s o f a u n i t w hich c o n s i s t s o f th e f i r s t

11 n u c le o t id e s o f th e s t r a i n 17 12 n u c le o t id e r e p e a te d u n i t .

A d ja cen t to t h i s a re 6 c o p ie s o f a u n it w hich c o m p r ises th e

- 120 -

I I n u c le o t id e s w ith the seq u en ce TCTGTGGGTGGGG in s e r t e d

a f t e r th e th ir d n u c le o t id e . R e i t e r a t io n I I I was a l s o

i d e n t i f i e d in s t r a in USA-8, but th e seq u en ce was n o t

a n a ly se d in d e t a i l .

A p a r t i a l o v e r la p o f th e r e p e a te d u n i t o f r e i t e r a t i o n s

I , I I and I I I i s p r e s e n t im m ed ia te ly to th e r ig h t o f ea ch

r e i t e r a t i o n d e f in e d by th e b r a c k e ts in P ig u r e C 3 . l l . The

lo c a t io n o f ea ch p a r t i a l o v e r la p i s a r b it r a r y and depends

on th e d e f i n i t i o n o f ea ch r e p e a te d u n i t . The p a r t ia l

o v e r la p s c o n s i s t o f th e f i r s t 7 , H and 10 n u c le o t id e s o f

th e r e p e a te d u n i t s o f r e i t e r a t i o n s I , I I and I I I r e s p e c t i v e l y .

No tandem r e i t e r a t i o n s e q u iv a le n t to th o s e p r e s e n t in

th e HSV-1 a seq u en ce were found in th e th e HSV-2 seq u en ce

(P ig u r e C 3 . l l ) . I t i s n o t known w h eth er r e i t e r a t i o n s

e q u iv a le n t to I I and I I I e x i s t i n HSV-2 DNA.

Com parison o f n u c le o t id e seq u en ces

The r e g io n o f g r e a t e s t d i f f e r e n c e i n th e j o i n t s eq u e n c es

o f HSV-1 s t r a i n s 17 and USA-8 i s in th e c seq u en ce im m ed ia te ly

a d ja c e n t to th e a se q u e n c e . The d i f f e r e n c e s in t h i s r e g io n ,

w hich i s AC r ic h on th e s tra n d shown in P ig u re C 3 * l l ,

r e p r e s e n t both s u b s t i t u t io n o f n u c le o t id e s and d e l e t io n o r

in s e r t io n o f s h o r t s e q u e n c e s . Only one n u c le o t id e d i f f e r s

betw een th e 310 a n a ly se d n u c le o t id e s o f th e b se q u e n c e s to

th e l e f t o f th e f i r s t 17 n u c le o t id e r e p e a t a t 3 1 1 -3 2 7 , and

s t r a in s 17 and USA-8 a re i d e n t i c a l i n 166 com parable n u c le o t id e s

a t th e r ig h t hand end o f th e c seq u en ce shown (1 6 0 1 -1 7 6 6 ) .

R e it e r a t io n I i s th e r e g io n o f g r e a t e s t d i f f e r e n c e betw een

th e a se q u e n c e s o f th e two HSV-1 s t r a i n s , and th e r e a re

ten b ase s u b s t i t u t io n s and one d e le t e d n u c le o t id e in th e

rem ainder o f th e a se q u e n c e . S in c e r e i t e r a t i o n s I , I I and

I I I were i d e n t i f i e d in both s tr a in s * a lth o u g h th e s e d i f f e r

- 121 -

w id e ly in tim e and geography o f i s o l a t i o n , i t seem s l i k e l y

th a t th e y w i l l be a g e n e r a l f e a tu r e o f HSV-1 i s o l a t e s .

The a se q u e n c e s o f HSV-1 and HSV-2 b oth h ave a h ig h

GC c o n te n t (835* and 845* r e s p e c t i v e l y ) , but th e seq u e n c es f . A

show l i t t l e s i m i l a r i t y . Y /ith in th e a se q u e n c e s th e lo n g e s t

s im i la r s e q u e n c e , w hich i s a l s o h ig h ly asym m etric betw een

th e two s tr a n d s o f th e d u p lex , i s 3 6 2 -2 8 9 o f HSV-1 s t r a in

17 and 9 4 -1 2 3 o f HSV-2 s t r a in HG52. U n lik e HSV-1, HSV-2

d o es n o t p o s s e s s r e i t e r a t i o n I , but b oth s e r o ty p e s have a

d ir e c t r e p e a t a t th e b -a and a - c j u n c t io n s .

B x p r e ss io n o f th e HSV-1 j o i n t r e g io n

There i s a s y e t no e v id e n c e t h a t t r a n s c r ip t io n s t a r t s

in th e n u c le o t id e seq u en ce o f th e HSV-1 j o i n t r e g io n shown

in P ig u re 0 3 .1 1 , and no. seq u e n c es c o n s i s t e n t w ith th e

c o n sen su s "H ogness box" (Gannon e t a l • , 1 9 7 9 ) a re p r e s e n t .

The h e x a n u c le o t id e AAUAAA has been fou nd i n c l o s e p r o x im ity

to th e 3' te r m in i o f th e m a jo r ity o f p o ly a d e n y la te d

e u k a r y o t ic mRNAs y e t a n a ly se d (B e n o is t e t a l . » 1 9 8 0 ) . A

few e x c e p t io n s , w hich p o s s e s s a seq u en ce c l o s e l y r e la t e d

to AAUAAA, have been n o ted (MacDonald e t a l * , 1980;

Hobart e t a l . , 1980; G oeddel e t a l . , 1 9 8 1 ) . The DNA seq u en ce

AATAAA i s lo c a t e d a t two p o s i t i o n s in th e s t r a in 17r j o in t

r e g io n , a t 1 5 0 0 -1 5 0 5 and on th e o p p o s i t e s tr a n d a t 1 5 4 6 -1 5 4 1 .

B e n o is t e t a l . (1 9 8 0 ) have o b serv ed a c o n ser v e d -sequence/ T T ~ T C . ’ T C C

r e la t e d to TTTTCACTGC (more a c c u r a t e ly g$ t gaa *at$ )

lo c a t e d 3 1 to th e AATAAA c lo s e to th e p o ly a d e n y la t io n s i t e

o f s e v e r a l , but n o t a l l , mRNAs, and th e r e la t e d seq u en ce

TTTGCAGTAG i s p r e s e n t a t 1 5 2 0 -1 5 1 1 3 ’ to th e AATAAA a t

1 5 46-1541 ( c o n s id e r in g th e o th e r s tr a n d o f th e d u p lex from

th a t show n). No such seq u en ce i s p r e s e n t 3* to th e o th e r

- 122 -

AATAAA.

These r e s u l t s s u g g e s t th a t a mRNA i s t r a n s c r ib e d from

r ig h t t o l e f t and i s p o ly a d e n y la te d in th e r e g io n 1 5 3 0 -1 5 2 0 .

E xp erim en ts were c a r r ie d o u t in c o l la b o r a t io n w ith Dr F .J .

R ixon to a s c e r t a in w hether t h i s i s in d e e d th e c a s e . The

Tag I D fragm en t (F ig u r e s C 3.3 and C 3 .9 ) , c o n s i s t in g o f

n u c le o t id e s 1 4 9 8 -1 6 0 8 , was l a b e l l e d a t th e 3' t e r m in i by32in c o r p o r a t io n o f o<- P-dCTP u s in g th e Klenow fragm en t o f

E . c o l i DNA p o ly m e r a se . The two s tr a n d s were s e p a r a te d by

p o ly a c r y la m id e g e l e l e c t r o p h o r e s i s under d e n a tu r in g c o n d it io n s

(8 M u r e a ) . H S V - l- in fe c te d c e l l mRNA was produced under

im m ediate e a r ly c o n d it io n s (c y c lo h e x im id e b lo c k ; C lem ents

£ £ 1 9 7 7 ) , from w hich th e 4*7 kb mRNA was i s o l a t e d from

m eth y lm ercu rie h y d ro x id e a g a r o se g e l s (W atson e t a l . , 1 9 7 9 ) .

A liq u o ts o f e i t h e r t o t a l im m ediate e a r ly RNA or th e 4*7 kb

mRNA were h y b r id is e d s e p a r a te ly w ith th e two u n iq u e ly -

l a b e l l e d DNA s in g le s tr a n d s (5 7 ° in 3 x SSC and 9 0 #^ 4

form am ide f o r 5 h r) a c c o r d in g to th e method o f Berk and

Sharp (1 9 7 7 ) . The sam p les were t r e a te d w ith S I n u c le a s e

to d egrad e s in g le - s t r a n d e d n u c le ic a c id s , and th e n s u b je c te d

to p o ly a c r y la m id e g e l e l e c t r o p h o r e s i s under d e n a tu r in g

c o n d it io n s (8 M u r e a ) . Only one o f th e DNA s tr a n d s was

p r o te c te d by RIM A, so t h i s was compared w ith th e n u c le o t id e

seq u en ce o f th a t fragm en t on th e same g e l (P ig u r e C 3 # l6 ) .

T h is r e s u l t shows t h a t th e 3 1 te r m in a l coded n u c le o t id e o f

th e mRNA c o d in g f o r Vmw IE 175 i s lo c a t e d i n th e r e g io n

1 5 3 0 -1 5 2 5 . No t r a n s c r ip t s from th e o th e r s tr a n d te r m in a t in g

c l o s e to th e AATAAA a t 1 5 0 0 -1 5 0 5 were d e t e c t e d , e i t h e r i n

im m ediate e a r ly RNA o r in RNA i s o l a t e d from c e l l s h a r v e s te d

6 hr p o s t i n f e c t i o n a t 3 7 °•

I t i s n o t y e t known w hether j o in t se q u e n c e s o th e r

- 123 -

than th o s e c o d in g f o r Vmw IE 175 mRNA a re r e p r e s e n te d in

s t a b le t r a n s c r i p t s , o r where prim ary t r a n s c r ip t io n t e r m in a te s .

The r e g io n c o n t a in s s e v e r a l p o t e n t i a l s p l i c e donor (PuGTXXG)

and a c c e p to r (PyPyXPyAG) s ig n a l s ( S e i f e t a l . , 1 9 7 9 ) , but

th e s e ca n n o t be in t e r p r e t e d in th e a b se n c e o f t r a n s c r ip t

mapping d a ta . Three f e a t u r e s o f th e DNA seq u en ce make i t

l i k e l y t h a t a c o n s id e r a b le p a r t o f th e j o i n t r e g io n d o es n o t

code f o r p o ly p e p t id e , n o ta b ly th e g and a d ja o e n t c s e q u e n c e s .

F i r s t , t r a n s la t io n i s u n l ik e ly to o ccu r th rou gh r e i t e r a t i o n I

in th e a se q u e n c e . V a r ia t io n in th e copy number o f th e 11

n u c le o t id e r e p e a te d u n i t o f t h i s r e i t e r a t i o n in s t r a i n USA-8

co u ld r e s u l t in t r a n s l a t io n o f ea ch o f th e th r e e p o s s ib le

downstream r e a d in g fr a m e s . S im ila r argum ents a p p ly to th e

c seq u en ce a d ja c e n t to a , w hich c o n t a in s r e i t e r a t i o n s I I and

I I I . S econ d , i f t r a n s la t io n o f mRNA r e p r e s e n t in g r e i t e r a t i o n

I o f s t r a in 17 o c c u r r e d , th e p o ly p e p t id e would c o n ta in th e

u n u su a l s t r u c t u r e o f 18 tandem r e p e a t s o f 4 amino a c id s

(p la sm id k l ) . S im i la r ly , r e i t e r a t i o n I I I would code f o r a

p o ly p e p t id e c o n ta in in g 6 tandem r e p e a t s o f 17 amino a c i d s .

T hird , th e a b se n c e o f n u c le o t id e s from one s t r a in compared

w ith th e o th e r would g e n e r a te t r a n s l a t io n a l f r a m e s h i f t s .

S tr a in USA-8 h as one n u c le o t id e in s e r t e d betw een 328 and

329 o f s t r a in 1 7 , one n u c le o t id e d e le t e d a t 3 4 0 , and f i v e

n u c le o t id e s in s e r t e d betw een 740 and 741 w hich would produce

t h i s e f f e c t .

- 124 -

DISCUSSION

F e a tu r e s o f r e i t e r a t i o n s

D eterm in a tio n o f th e DuA seq u e n c es a t th e j o in t r e g io n

betw een th e 1 and 3 segm en ts o f th e genom es o f HSV-1 and HSV-2

h as r e v e a le d s e v e r a l f e a t u r e s . The p r e se n c e o f r e i t e r a t i o n s i n

HSV-1 p r o v id e s a read y e x p la n a t io n f o r th e w id e ly o b serv ed s i z e

v a r i a b i l i t y in th e a and c s e q u e n c e s . The HSV genome i s known

r e a d i ly to undergo reco m b in a tio n d u r in g r e p l i c a t i o n , and u n eq u a l

c r o s s o v e r e v e n ts oetw een tandem r e i t e r a t i o n s would g iv e r i s e to

s i z e v a r i a b i l i t y in th e s e r e g io n s . S im ila r tandem r e i t e r a t i o n s

m ight be r e s p o n s ib le f o r such v a r i a b i l i t y e lse w h e r e i n th e

genome. A f u n c t io n a l r o le f o r th e r e i t e r a t i o n s h a s n o t y e t

been i d e n t i f i e d , and i t i s p o s s ib le t h a t th e y encode no

e s s e n t i a l f u n c t io n s but r e p r e s e n t r e g io n s in w hich e r r o r s in

DNA r e p l i c a t i o n can a ccu m u la te . The a b se n c e o f r e i t e r a t i o n I

in th e a seq u en ce o f HSV-2 s t r a in HG52 a r g u e s a g a in s t an

e s s e n t i a l r o l e f o r th a t tandem r e i t e r a t i o n i n v ir u s

r e p l i c a t i o n . The 11 n u c le o t id e s CCCGCTCCTCC w hich form th e

f i r s t r e p e a te d u n i t o f r e i t e r a t i o n I in HSV-1 s t r a i n USA-8

( in t h i s c a s e r e d e f in in g th e u n it shown i n H g u r e C 3*H by

m oving th e b r a c k e ts one n u c le o t id e to th e l e f t ) a re a l s o

p r e s e n t a t 8 7 1 -8 8 1 (w ith r e s p e c t to th e s t r a i n 17 s e q u e n c e ) ,

show ing t h a t t h i s seq u en ce i s n o t a m p lif ie d o f n e c e s s i t y i n

th e HSV-1 genom e. The r e p e a te d u n i t s o f th e r e i t e r a t i o n s ,

a lth o u g h d i f f e r e n t , have s i m i l a r i t i e s such a s a p rep on d eran ce

o f C r e s id u e s on one s tra n d o f th e d u p lex and G r e s id u e s on

th e o th e r , and i t i s p o s s ib le t h a t such se q u e n c e s a r e more

r e a d i ly a m p lif ie d th an o t h e r s . T h is s im i l a r i t y ex te n d s a l s o

to r e i t e r a t i o n s IV and V w hich a re lo c a t e d e lse w h e r e i n th e

iiSV-1 genome, a s sum m arised in F ig u r e C 3 .1 8 . I t i s o f

in t e r e s t ' to n o t e t h a t th e seq u en ce CPyCCXCCC i s p r e s e n t in

- 125 -

r e i t e r a t i o n s I and I I I o f both HSV-1 s t r a i n s , and in

r e i t e r a t i o n IV; in d eed i t i s p r e s e n t on b oth s tr a n d s o f th e

24 n u c le o t id e r e p e a te d u n i t o f r e i t e r a t i o n I i n s t r a i n USA-8 .

GGCCTCCC i s p r e s e n t in r e i t e r a t i o n I I and CCCCACTC (CPyCCXCTC)

i n r e i t e r a t i o n V. The SV40 genome p o s s e s s e s s i x c o p ie s o f

PyPyCCGCCC c l o s e to th e o r ig in o f DNA r e p l i c a t i o n (Dhar e t

a l . , 1 9 7 7 ) , two o f w hich a re w ith in T -a n t ig e n b in d in g s i t e

I I I ( T j ia n , 1 9 7 8 ) . However, th e p r e se n c e o f t h i s b in d in g s i t e

i s n o t e s s e n t i a l f o r SV40 DNA r e p l i c a t io n (Subram anian and

Shenk, 1 9 7 8 ) . T -a n t ig e n b in d in g s i t e I I , . th e p r e se n c e o f w hich

i s r e q u ir e d f o r DNA r e p l i c a t io n (Shenk , 1 9 78; Gluzman e t a l . ,

1 9 8 0 ) , p o s s e s s e s two p a lin d ro m es o f GCCTC (T j ia n , 1 9 7 8 ) , and

r e i t e r a t i o n s I -I V c o n ta in PyCCTC and r e i t e r a t i o n V c o n ta in s

CACTC. Such se q u e n c e s , • how ever, a re s u f f i c i e n t l y s h o r t to be

p r e s e n t commonly i n HSV-1 DNA ( e . g . PyPyCCGCCC a t 1 6 8 8 -1 6 9 5

and GCCTC a t 1 6 8 6 -1 6 9 0 ) , and so i t rem ain s to be shown t h a t

th e se q u e n c e s o f th e r e p e a te d u n i t s a re s p e c i f i c f o r

p r e f e r e n t i a l a m p l i f i c a t io n .

C oding p o t e n t i a l a t th e 3* end o f Vmw IE 175 mRNA

The g en es co d in g f o r dPyK (M cK night, 1 9 8 0 ) , Vmw IE 175

( t h i s s tu d y ; M-J. M urchie, p e r s o n a l co m m u n ica tio n ), Vmw IE 12

(M -J. M urchie and F .J . R ixon , p e r s o n a l com m u n ication s) and

Vffiw 1 3 6 ’ (1 4 3 ) (J . M cLauchlan, p e r s o n a l com m unication) have

5 ! and 3 ’ seq u e n c es c o n s i s t e n t w ith co n serv ed f e a t u r e s o f

e q u iv a le n t r e g io n s o f o th e r e u k a r y o t ic g en es c o d in g f o r

p o ly a d e n y la te d mRNAs. The o n ly in t r o n s y e t d e s c r ib e d f o r HSV-1

r e s id e in TR5/I R 3 in th e g en es co d in g f o r Vmw IE 12 and 68

(Y/atson e t a l . , 1981; P .J . R ixon and M-J. M urchie, p e r s o n a l

0 0JU:i»Ui xca ijiuhs) .

The seq uence a t th e 3 1 term in u s o f th e Vmvv IE 175 mRNA

(P ig u re C 3 . l l ) c o n ta in s two p o s s ib le te r m in a t io n cod on s in

- 126 -

d i f f e r e n t fra m es, a t p o s i t i o n s c o r r e sp o n d in g to 156 2 -1 5 6 0

(UGA) and 1 5 9 1 -1 5 8 9 (UAA). The h y p o t h e t ic a l amino a c id

seq u en ce o f th e carb oxy term in u s o f Vmw IE 175 u t i l i s i n g each

o f t h e s e te r m in a t io n codons i s g iv e n i n T ab le C% 6. A lso

shown i s th e o c cu r r en ce in each c a s e o f th e fo u r amino a c id s

coded by t r i p l e t s c o n ta in in g c o m b in a tio n s o f G and C m o ie t ie s

o n ly , and th e d i s t r i b u t i o n o f th e fo u r n u c le o t id e s in th e

th ir d codon p o s i t i o n . Two o b s e r v a t io n s from t h e s e d a ta

s u g g e s t t h a t , i f e i t h e r te r m in a t io n codon i s u se d , th e UAA

fram e i s th e more l i k e l y . F i r s t , t h i s fram e c o n ta in s f a r

few er a r g in in e r e s id u e s in t h i s r e g io n and t h e r e fo r e m ight be

th ou gh t to code f o r a more r e a s o n a b le p r o t e in s t r u c t u r e .

S econd , th e g r e a t m a jo r ity o f cod on s i n t h i s fram e have G or

C r e s id u e s in th e th ir d p o s i t i o n . T h is i s c o n s i s t e n t w ith

th e e x p e c t a t io n th a t t h i s r e g io n o f th e DNA would p o s s e s s a

h ig h GC c o n te n t e s p e c i a l l y i n th e t h ir d codon p o s i t i o n , s in c e

i t i s under s e l e c t i v e p r e s s u r e s to m a in ta in b oth a h ig h GC

c o n te n t ( f o r unknown r e a s o n s ) and a f u n c t io n a l Vmw IE 175 g e n e .

F u l l e lu c id a t io n o f th e c o d in g r e g io n o f t h i s gene a w a it s

co m p le te seq u en ce d e te r m in a t io n .

R ole o f th e a seq u en ce in c o d in g f o r p o ly p e p t id e

I t seem s l i k e l y t h a t th e HSV-1 a seq u en ce d o es n o t code

f o r p o ly p e p t id e . P r e v io u s ly , th e l e s i o n in an HSV-1 t s

mutant ( t s c 7 5 ) had been i n c o r r e c t ly mapped i n th e a seq u e n c e ,

and ex a m in a tio n o f th e p o ly p e p t id e s in d u ced by t h i s m utant a t

th e n o n -p e r u .is s iv e tem p era tu re prompted th e c o n c lu s io n t h a t th e

a seq u en ce form s p a r t o f th e Vmw IE 175 gen e (K nipe e t a l . , 1 9 7 9 ) ,

T h is s tu d y shows t h a t th e a seq u en ce i s n o t in c lu d e d i n th e

g en e . P r e s to n ( in p r e s s ) h as shown t h a t th e m u ta tio n maps

in th e c seq u en ce some 2 kbp away from th e a se q u e n c e . T hese

f in d in g s have r e p e r c u s s io n s d e tr im e n ta l to th e h y p o th e s is t h a t

- 127 -

a l l a s e q u e n c e s w ith in a genome a r e o b l i g a t o r i l y i d e n t i c a l ,

a s d is c u s s e d i n S e c t io n 4 .

Homology betw een th e j o i n t r eg io n s o f th e HSV-1 and HSV-2 genom es

H y b r id is a t io n ex p er im en ts (F ig u r e C 3 .7 ) d em on stra ted

hom ology betw een HSV-1 BamHI k and th e c seq u en ce o f HSV-2

BamHI g , and betw een HSV-2 BamHI g and th e c seq u en ce o f HSV-1

BamHI k . These r e g io n s co rresp o n d i n l o c a t io n to th e 3 !

p o r t io n s o f th e g e n e s c o d in g f o r HSV-1 V IE 175 andmwHSV-2 Vffiw IE 1 8 2 . A more l o c a l i s e d r e g io n o f hom ology i n th e

b seq u e n c es (HSV-1 Smal H and HSV-2 Smal 4 ) was a ls o

d e t e c t e d . I t was n o t shown th a t hom ology e x i s t s betw een th e s e

two fr a g m e n ts , a lth o u g h t h i s i s th e s im p le s t e x p la n a t io n o f

th e r e s u l t s . HSV-1 Smal H i s a d ja c e n t to se q u e n c e s c o d in g f o r

th e 5 1 term in u s o f Vmw IE 110 mRNA (Mackem and Roizm an, 1 9 8 0 ) ,

and t h e r e f o r e i t i s p o s s ib l e t h a t HSV-1 and HSV-2 sh are

hom ology in t r a n s c r ip t io n a l c o n t r o l seq u e n c e s a t th e 5 1

te r m in i o f th e g en es co d in g f o r Vmw IE 110 (HSV-1) and

\ w IE 118 (H SV -2). Fragm ents c o n ta in in g th e a seq u en ce d id

n o t h y b r id is e w e l l even to hom ologous p r o b e , and i t was

th e r e fo r e n o t p o s s ib le to e s t im a te th e e x t e n t o f hom ology

betw een th e HSV-1 and IISY-2 a se q u e n c e s .

R ole o f th e a seq u en ce in m a tu ra tio n o f genome le n g t h DNA

In both iISV-1 and HSV-2 th e S term in u s i s e q u iv a le n t

to a p o s i t i o n c lo s e to th e 'r ig h t band end o f th e 17 and 21

n u c le o t id e d i r e c t r e p e a t a t th e b -a j u n c t io n , and th e L

term in u s i s e q u iv a le n t to th e r ig h t hand end o f th e o th e r

d i r e c t r e p e a t a t th e a -c ju n c t io n . A lth ough th e lo c a t io n s o f

th e te r m in i w ith r e s p e c t to th e j o in t seq u en ce have n o t been

d e f in e d to s in g le n u c le o t id e s , F ig u r e 03*11 shows t h a t th e

HSV-1 s t r a in USA-8 S term in u s i s one n u c le o t id e to th e r ig h t

o f t h a t i n ..SV -l s t r a in 1 7 , w hereas th e L te r m in i c o in c id e .

- 128 -

A m in o r ity o f v i r io n UNA m o le c u le s c o n ta in more than one copy o f

th e a seq u en ce a t th e 1 term in u s and a t th e j o i n t . D e te r m in a tio n

o f th e n u c le o t id e seq u e n c es betw een two tandem IISV-l o r HSV-2

a seq u e n c e s h as shown th a t th e a se q u e n c es a r e arran ged a s

h e a d - t o - t a i l d im ers w hich have one copy o f th e d i r e c t r e p e a t

a t t h e i r p o in t o f f u s io n (P ig u r e 0 3 .1 7 )* T h is arrangem ent

c o u ld a r i s e e i t h e r by m is a lig n e d g e n e t i c re co m b in a tio n a t th e

d i r e c t r e p e a t s , o r by d i r e c t l i g a t i o n o f th e L and S te r m in i*

The f a c t t h a t m u lt ip le a se q u e n c es have n o t been o b se rv ed a t

th e S term in u s o f HSV-1 s t r a i n 17 and HSV-2 s t r a in HG52

presum ably r e f l e c t s th e m echanism o f DNA r e p l i c a t i o n and

m a tu r a tio n . I t i s p o s s ib le t h a t th e d i r e c t r e p e a t s s e r v e a s

r e c o g n i t io n s i t e s f o r th e m a tu ra tio n o f u n i t l e n g th DNA

from co n ca tera ers . S in c e th e HSV genom ic t e r m in i a r e ’

s p e c i f i c a l l y lo c a t e d , i t i s l i k e l y t h a t c o n c a te m e r ic DNA i s

s p e c i f i c a l l y c le a v e d a t a l t e r n a t e j o i n t r e g io n s to produce

DNA o f genome l e n g t h , b u t th e r e i s a s y e t i n s u f f i c i e n t e v id e n c e

upon w h ich to b ase d e t a i l e d m o d e ls . N e v e r t h e le s s , th e

co n seq u en ce o f th e m a tu ra tio n m echanism i s t h a t th e L term in u s

e s s e n t i a l l y c o n ta in s b oth c o p ie s o f th e d i r e c t r e p e a t , w h ereas

th e S term in u s c o n t a in s o n ly th e a -c copy (P ig u r e C 3 .1 7 ) .

R ole o f th e a seq u en ce i n segm ent in v e r s io n

D ata w hich s u g g e s t t h a t th e a seq u en ce h a s a r o l e i n

segm ent in v e r s io n by a s i t e - s p e c i f i c m echanism a re d is c u s s e d i n

S e c t io n 4 . P r e c i s e ly w hich se q u e n c es a r e in v o lv e d in th e

p rop osed s i t e - s p e c i f i c reco m b in a tio n e v e n ts i s unknown. One

p o s s i b i l i t y i s t h a t th e r e g io n w hich i s co n serv ed betw een th e

HSV-1 and HSV-2 a seq u en ces (3 6 2 -3 8 9 in P ig u r e C3*HA and

9 4 -1 2 3 -in P ig u re C 3.11B) i s a reco m b in a to r seq u en ce a n a lo g o u s

to c h i in p r o k a r y o te s (Sm ith e t a l . , 1980) and co g in fu n g i

(C a tc h e s id e and C orcoran , 1 9 7 3 ) , p rom otin g h ig h l o c a l r a t e s o f9

- 129 -

recom bination, or that i t i s equivalent to a t t in lambda

(Dandy and Ross, 1977)> promoting recombination in a sp e c if ic sequence. These hypotheses could be te s te d by tran sferrin g

th is sequence to any other rea d ily s t u d i e d genome. Purther

aspeots o f the ro le o f the a sequence in DNA r e p lic a t io n are

discussed in Section 4-

Table 03*1

S iz e s (bp) o f r e s t r i c t i o n fra g m en ts o f HSV-1 BamH I k

and 11SV-2 BamH I g . These fra g m en ts c o n ta in one a

seq u e n ce and , s in c e th e r e a r e r e g io n s o f s i z e

v a r i a b i l i t y i n HSV-1 BamH I k , s i z e s r e f e r

s p e c i f i c a l l y to th e recom b in an t p la sm id k l 3hown

in P ig u re 0 3 * 6 , T o ta l s i z e s o f th e fra g m en ts

a re g iv e n , and r e s t r i c t i o n maps a r e shown in

P ig u re 0 3 .3

Table C 3.1

IiSV-1 BamH I k

S g t I Sma I

A 3500 A 1100

B 1600 B 840

C 510 C 700

G 330 D. 560

£ 560

F ' 410

G 340

H 350

I 275

i 240

K 150

L 143

Ivl 91

N 86

0 48

P 43

Q 38

R 24

5940 6015"

HSV-2 Bamii I £

s s i x Sma I

1 2020 1 2650

■2 1310 2 1050

3 960 3 540

4 740 4 510

5 390 5 440

6 290 6 300

7 160 7 112

8 130 8 87

9 76

10 67

11 63

12 14

6000 5909

Table 03.2

D ata f o r th e r e s t r i c t i o n mapping o f HSV-1 BamHI k .

M ost e x p e r im e n ts u sed t h i s fragm en t i s o l a t e d from

p la sm id k l (F ig u r e 0 3 . 6 ) , e x c e p t in p a r t s B, G, F

and G, when th e fra g m en ts were i s o l a t e d from v ir io n

DBA. N ick t r a n s la t e d DNA was u sed e x c e p t i n p a r t H,

when a fragm en t ^2P - la b e l l e d a t th e 5 f te r m in i was

u s e d . Fragm ents i n p a r e n th e s e s were n o t r e s o lv e d ,

and so in d ic a t e p r e d ic t e d r a th e r th an a c t u a l s i z e s .

E le c t r o p h o r e s is was c a r r ie d o u t on 2 # a g a r o s e ,

7 .5 ,‘* p o ly a c r y la m id e o r 15/^ p o ly a c r y la m id e g e ls *- *

A garose g e l s c o n s i s t e n t l y gave la r g e r v a lu e s f o r

s i z e s o f fr a g m en ts g r e a t e r th a n 500 bp than

p o ly a c r y la m id e g e l s . T h ere fo re s i z e s above 500 bp

a re q u oted from a g a r o se g e l s , and below 500 bp from

p o ly a c r y la m id e g e l s . T he.deduced r e s t r i c t i o n maps

a re shown i n F ig u re C3*3*

Table 05.2

A. Fragment s i z e s (bp) o f HSV-1 BamH I k .

S s t I 3500 1600 510 330

Sau3A I 3300 1600 900 170

Tag. I 3700 1270 900 110 26

H inc I I 4600 1400

Sma I 1100 840 700 560 560 410 340 350 275240 150 143 91 86 48 43 38 24

B. Fragment s i z e s (bp) o f HSV-1 BamH I q .

S s t I 2700 510 330

Sma I 1100 840 560 410 340 91 48 3b 37 24

C. Fragment s i z e s (bp) o f HSV-l BamH I s ,

S s t I 1600 1300

Sma I 700 560 350 340 275 240 150 143 86 43

D. Fragment s i z e s (bp) o f d ou b le d i g e s t s o f HSV-1 BamH I k .

Sma l/S s_ t 1 1100 760 700 560 410 35Q 340 275260 250 240 150 143 91 86 78 70(48 43 38 2 4 )

Sma 1 / Sau3A I 700 660 560 560 510 420 410 350 340275 240 170 150 150 143 91 86(48 43 38 2 4 )

Sma I/Tag. I 820 680 700 560 560 410 350 340 275240 150 150 143 140 110 91 86 4338 26 24 22

Sma i / l l i n c I I 1100 700 680 560 560 410 350 340275 240 160 150 143 91 86 48 4338 24 V

S s t l/S a u 3 A I 1700 1600 1600 510 330 170 70

S s t I/Tag. I ( 200u 1600 / 1270) 510 300 110 70 26

S s t I /H in c I I 2900 1600 6O0 510 330

Sau3A I /T a q 1 (3300 1100 900) 400 170 1J0 26

Table 03.2

£ . fra g m en t s i z e s (bp) o f d ig e s t s o f fra g m en ts o f HSV-1 BamH I k .

fragm en t d ig e s t e d w ith p ro d u cts

S s t I A Sma I 1100 720 410 350 150 80 43 24

S s t I B Sma I 700 560 240 143

S s t I C Sma I 260 91 78 38

S s t I £ * Sma I 250 48

Sau3A I A Sma I 700 560 430 350 240 150 86 43

Sau3A I B Sma I 640 495 410 24

Sau3A I C Sma I 560 150 91 48 38

S s t I A Sau3A I (1650 8 4 0 ) 560 75

Sst; I C Sau3A I 510

S s t I A Tag 1 (2000 8 4 0 ) HO 70

S s t I C Tag. I 510

Sau3A I A Tag I (3 3 0 0 )

Sau3A 1 B Taa I (1 1 0 0 ) 350 110

P. Pragiaent s i z e s (bp) o f d i g e s t s o f fra g m en ts o f 1I3V-1 BamLi I cl# -

f ragm ent

S s t I A

S s t I C

S s t I 1)

d ig e s t e d , w ith

Sma I

Sma I

Sma I

p r o d u c ts

1100 '720 410 340 40 24

260 91 78 38

250 48

Table 03.2

G. Ifragment s i z e s (bp) o f d i g e s t s o f fragments? o f HSV-1 Bamil I s . .

fragm en t dig e s t e d w ith produc t s

Sma I 700 560 240 143

Sma I 350 340 275 150 80 43

H. Sma I p a r t i a l d ig e s t io n p r o d u c ts (bp) from ea ch o f th e two h a lv e s o f HSv-1 BamH I k Sau3A I k , w hich had been 5 ' - l a b e l e d a t th e Sau3A I sltes*~and tEe two h a l v e s ’ s e p a r a te d by d ig e s t io n w ith S s t I , and t h e ir in t e r p r e t a t io n a s Sma I fr a g m e n ts .

l e f t hand fragm en t o f 5 a u 3 r r r ---------------

240 (J)

950 (J-C)

1100 (J-C -L )

1650 (J -C -L -h )

I . Sma I p a r t i a l d ig e s t io n p r o d u c ts (bp) o f HSV-1 .BamH I k Sau3A I B w hich would be p r e d ic te d from th e tw o ~ p o s s ib le a rra n g em en ts .

f ragm ent o rd er pr e d ic t e d p a r t i a l d ig e s t io n p ro d u cts

640-K -JM 95 1 H 5 970 945 665 475

640-F -R -495 1115 i09Q 970 520 475

r ig h t hand fragm en t o f'S e f i S r T T . -------------

430 (p a r t o f A)

770 (4 3 0 -£ )

820 (430-G -P )

1 0 0 0 * (430-G -P -K )

1290 ( 430-G -P-K -X )

1630 ( 430-G -P -iC -I-H )

1700 (430—G -P -K -I-H -p a rt o f N)

S s t I £

S s t X X

Table 03.2

J . Sma I p a r t i a l d i g e s t i o n p r o d u c ts (bp) o f HSV-1 BamH I k S s t I C w hich would be p r e d ic te d , from tEe two“ p o s s i b l e a rra n g em en ts .

fragm en t o r d e r p r e d ic t e d p a r t ia l d i g e s t i o n p ro d u cts

78—M—(J—260 389 298 207 169 129

7 8-£}-M-2 60 389 351 207 129 116

Table C3.3

B ata f o r th e r e s t r i c t i o n mapping o f fiSV-2 Bamiil g .

M ost e x p er im e n ts u sed t h i s fragm en t i s o l a t e d from

p la sm id g l (F ig u r e 0 3 . 6 ) , e x c e p t in p a r t s 3 , C, 3,

G and H, when th e fra g m en ts were i s o l a t e d from v ir io n

BNA. The p la sm id fragm en t c o n ta in s two tandem

r e p e a te d a seq u e n c e s in s t e a d o f th e s in g l e a seq u en ce

p r e s e n t in th e m ajor j o i n t fragm en t from v i r i o n BNA.

T h is e x t r a 250 bp r e s u l t s , f o r exam p le , in two

a d d i t io n a l fra g m en ts in . th e Smal d i g e s t w hich a re

n o t produced from th e norm al j o i n t (230 and 14 b p ).

T h is was ta k en i n t o a cco u n t in d ed u cin g th e

r e s t r i c t i o n maps shown in F ig u re C 3 .3 .

N ick t r a n s la t e d BNA was u se d , and fra g m en ts in

p a r e n th e se s in d ic a t e p r e d ic te d r a th e r than a c t u a l

s i z e s s in c e th e y were n o t r e s o lv e d on th e g e l u s e d .

E le c t r o p h o r e s is was c a r r ie d o u t on 25* a g a r o s e ,

7 .5 /* p o ly a c r y la m id e o r 155* p o ly a cr y la m id e g e l s .-

A garose g e l s c o n s i s t e n t l y gave la r g e r v a lu e s than

p o ly a c r y la m id e g e l s f o r s i z e s o f fra g m en ts g r e a te r

than 500 bp. T h ere fo re s i z e s above 500 bp are

q u oted from a g a r o se g e l s , and below 500 bp from

p o ly a c r y la m id e g e l s .

Table C3«3

A, Fragm ent s i z e s (bp,) o f HSV—2 BamH I w hich c o n ta in s two tandem ly r e p e a te d a s e q u e n c e s .

S s t I 2020 1550 960 740 390 290 160 130

Sau3A I 4100 1250 900

Tag I 1800 1600 1050 770 660 85 25

H inc I I 2600 1800 1300 250

Kffi I 4700 1500

Sma I 2650 1050 540 510 440 500 230 87 76 67 63 14 14

112

B, Fragment s i z e s (bp) o f HSV-2 BamH I u .

S s t I . 2020 390 390 290 130

Sma I 2650 440 87 22 14

0 . Fragment s i z e s (bp) o f HSV-2 BamH I v .

S s t I 1550 1200 960 160

Sma I 1050 540 510 300 215 112 76 67 6;

B. Fragment s i z e s (bp) o f d o u b le d i g e s t s o f HSV-2 BamH I g .

. •'* T'U■ WSma I / S s t I 2020 1000 540 460 390 350 . 290 230 ----- 210 112 87 87 76 67 63 62 40 23

Sma I/S au 3A I 1600 1050 900 540 460 440 300 230112 87 76 67 63 53

—Sma I/T ag. I 510 480 440 290 250 230 112 85 7667 63. 57 44 25

Sma I /H in c I I 1400 1100 1050 540 510 440 300 230112 87 76 67 63

Sma I/Krm I 1400 1100 1050 540 510 440 300 230112 87 76 67 63

Table C3.3

B. fragm en t s i z e s (bp) o f d o u b le d i g e s t s o f HSV-2 BamH I g

( c o n t in u e d ) .

S s t 1 / Sau3A 1 1600 1550 740 600 450 390 290 200160 130

— S s t i/T a a I 560 390 280 160 130 85 48 41 25

S s t I / H inc I I 1300 1000 960 740 460 390 360 290 250160 130

S s t I/K on I 1550 1050 960 750 740 390 290 160130— Sau3A I /H in c I I 290 250

— Sau3A I/K pn I 500

— Tac I /H in o I I 540 460 400 250 85 25

— Tag I/K pn I 85 25

- - H inc II/K pn I 2600 1800 1300 250 250

ita g m e n ts s m a lle r than 20 bp were n o t r e s o lv e d . For th o se d i g e s t s marked — fra g m en ts l a r g e r than 6Q0 bp were n o t r e s o lv e d .

IS. 'Fragment s i z e s (bp) o f d i g e s t s o f S s t I fra g m en ts o f HSV-2 BamH I g . .

fragm en t d ig e s t e d w ith p r o d u c ts

1 Sma I 2020

2 Sma I 1000 350 230

1 Sma I 460 290 112

1 Sma I 540 63

£ Sma I 390

6 Sma I 210 87

1 Sma I 76 62 23

8 Sma I 8 7 40

Table 05*3

E. fragm en t s i z e s (Jap) o f d i g e s t s o f S s t I fra g m en ts o f HSV-2 BamH I~ g ( c o n t in u e d ) . “

fragm en t d ig e s t e d w itb p r o d u c ts

1 Tag I (1050 1000)

2 Tag I (8 7 0 ) 530

1 Tdq I (9 2 0 ) 39

i Tag. I VJI o 00 VJI

Tag. I 390

6 Tag I 260 25

I Tag I 160

8 Tag I 130

Bo fragm en t s i z e s (bp) o f Sma I d i g e s t s o f S s t I fra g m en ts o f IISV-2 BamH - r B. —

S a t I 1 2020

S s t I 390 350 25 14

S s t I 6 210 8'7

S s t 1 8 87 40

0 . i'ragment s iz e s - (bp) o f Sma I d i g e s t s o f S s t I fra g m en ts o f HSV-2 BamH I v . ! -

S s t I X 1000 220 67 14

S s t I 2 460 290 112

S s t X 2 540 63

S s t I I 76 62 23

Table C?.3

H. fragm en t s i z e s (bp) o f Sma I p a r t i a l d i g e s t i o n o f S s t I fra g m e n ts , o f HSV-?"lkmH I g .

S s t I 1050 365 80

S s t I i 570 410

S s t I i 600 67

Table U3.4

i)a ta f o r th e r e s t r i c t i o n mapping o f th e 1760 bp

H in f I fragm en t w hich c o n ta in s th e j o i n t betw een HSV-1

1 and S . A lso u sed was th e Sau3A I /H in f I 1130 bp

fragm en t w hich sp a n s tn e j o i n t , and co m p r ise s th e

l e f t hand end o f th e 1760 bp fr a g m e n t•

K ick t r a n s la t e d DNA was p rep ared from recom b in an t

p lasm id k l (F ig u r e 0 3 . 6 ) , E le c t r o p h o r e s is was

c a r r ie d o u t on 2 y* a g a r o se o r 7*57* p o ly a c r y la m id e g e ls>« 4 s

and fragm en t s i z e s g r e a te r than 500 bp a re q uoted

from a g a r o se g e l s and l e s s than 500 bp from

p o ly a c r y la m id e g e l s . The deduced r e s t r i c t i o n maps

a re shown in F ig u re 0 3 .9 .

Table C?«4

A. fragm en t s i z e s (bp) o f th e HSV-1 JBamH I k Sau3A I /H in f I 1130 bp fra g m en t, w hich c o n t a in s tEe a se q u e n c e ,

Sma I 410 340 160 150 43

Ava I I 680 m

BstN I 1000 130

Ava I 360 340 160 150 50 43

Hae I I 980 150

B gl I 930 170 25

S s t I I 420 320 190 85 80 18

Tag. I 1130

U n d er lin ed fra g m en ts a r e th o se w hich were l a b e l l e d when th e 1130 bp fragm en t was p rep ared from 5* - l a b e l le d HSV-1 BamH I k Sau3A I 'A. ,

B. Pragment s i z e s (bp) o f th e HSV-1 BamH I k H in f I 1760 bp fra g m en t, w hich c o n t a in s th e a seq u e n c e .

Sma I 1040 340 160 150 43

Ava I I 1080 680

BstN I 1500 130 130

Ava I 680 350 340 160 150 43

Hae I I 1000 430 150 12 5 70

B £l I 1460 170 70 26 25

S s t I I 950 320 190 95 85 80 18

Tag I 1500 150 110

U n d erlin ed fra g m en ts were d e f i n i t e l y n o t p r e s e n t in th e H in f I d i g e s t o f n ic k t r a n s la t e d HSV-1 BamH I k .

C. Fragm ent s i z e s (bp) o f Bau4A I d o u b le d i g e s t s o f th e HSV-1 BamH I k H in f I 1760 bp fra g m en t.

Sau3A l / Sma I 630 410 340 160 150 43

Sau3A I / Ava I I 680 650 450

Sau3A I/BstIM I 1000 500 130 130

Sau3A I / Ava I 630 350 340 160 150 50 43

Sau3A I/H a e I I 980 430 150 125 70

Sau3A I / B gl I 930 530 170 70 26 25

Sau3A I / S s t I I 570 420 320 190 95 05 80 18

Sau3A I/T a g I 1130 370 150 110

D. Fragm ent s i z e s (b p) o f d o u b le d i g e s t s o f th e HSV-1BamH I k Sau3A I / H in f I 1130 bp fra g m en t.

Sma I / Ava I I

Sma I /B stN I

Sma I /H a e I I

Sma I /B g l I

Sma I / S s t I I

Ava I I /B s tN I

Ava I I /H a e I I

Ava I l / B g l I

Ava I I / S s t I I

BstN I /B g l I

BstN I / S s t I I

B g l I / Hae I I

B gl I / S s t I I

S s t I I / Ava I

S s t I I / f la e I I

B g l 1 / Ava I

410 310 160 150 45 43

410 340 150 130 43 32

410 340 150 150 43

410 340 160 125 43 27

260 130 65 80 58 - H CO

550 450 130

530 450 150

530 400 1'70 25

420 320 190 65 41 32 18

930 130 35 25

420 320 170 65 80 27 18

930 150 25 13

420 320 120 65 80 62 18

350 250 135 65 80 61 48

420 320 140 65 80 50 18

350 340 160 125 50 43 27

T a b l e 0 5 « 4

ii. ij'Tagment s i z e s (bp) o f d ou b le d i g e s t s o f HSV-1 BamH I k H in f I 1760 bp fra g m en t.

1 * 1 I /H ae I I 980 350 150 125 62 38 26

Tag. I /B g l I 1300 170 110 70 53 26 25

Tag I / S s t I I 800 320 190 110 95 85 60 50 18

m l I /H ae I I 930 430 150 70 70 29 26 25

Table 03.5

Bata f o r th e r e s t r i c t i o n mapping o f the 1310 bp HSV-2

S s t I fra g m en t w hich c o n ta in s bhe j o i n t betw een

L and S (HSV-2 BamH I & S s t I 2 in F ig u re 0 3 . 3 ) .

A lso u sed was th e Sma I 900 bp fragm en t w hich does'

n o t span th e j o i n t , b u t o v e r la p s a lm o st e n t i r e l y th e

1310 bp fragm en t a t i t s l e f t hand end (HSV-2 BamH I g

Sma I 2 in F ig u re C3o3J* N ick t r a n s la t e d BNA was

p rep ared from recom b in an t p la sm id g l (F ig u r e 0 3 ..6 ) '■

w hich c o n t a in s two tandem r e p e a te d a se q u e n c e s

in s t e a d o f th e one a seq u en ce p r e s e n t in th e m ajor

j o in t fra g m en t o f v i r i o n BNA, and so th e S s t I

fragm en t i s 1550 bp in t h i s c a s e ( s e e T ab le 0 3 . 3 ) .

T h is f a c t has been tak en in t o a cco u n t in d ed u c in g th e

r e s t r i c t i o n maps shown i n F ig u re 0 3 .9 .

E le c t r o p h o r e s is was c a r r ie d o u t on 2 °/o a g a r o se or 7 * 5 $

p o ly a c r y la m id e g e l s , and fragm en t s i z e s g r e a t e r4

than 500 bp a r e q u oted from a g a r o se g e l s and l e s s

than 500 bp from p o ly a c r y la m id e g e l s . The deduced

r e s t r i c t i o n mai)s a re shown in F ig u r e 03«9

Table 05.5

A. Fragment s i z e s (bp) o f HSV-2 BamH I g S s t I 2 , w hich c o n ta in s two tandem ly r e p e a te d a s e q u e n c e s .

Dde I 1200 360

A lu I 1440 125

H inc I I 960 550 250

Sma I 680 360 230 67 14 14

Ava I 500 360 280 230 130 67 14 14

Ava I I 830 550 100 70 27

BstN I 1000 250 170 70 29 15

S s t I I 480 540 310 210 200 28 28 14 14

H in f I 600 360 300 220 29 29 26

B. fragm en t s i z e s (bp) < HSV-2 BamH I g S s t I

o f Sma I 2 .

d ou b le d i g e s t s o f

Dde I 500 360 360 230 67

A lu I 880 240 230 125 67

Hinc II 880 360 230 67

BstN I 360 330 250 230 170 70 67 29

S s t I I 540 310 290 200 110 110 78 67 28

H in f I 600 360. 250 210 44 26 25 25

Fragm ents sm a lle r than 20 bp were n o t r e s o lv e d .

B ata c o n c e r n in g th e p o s s ib le amino a c id seq u e n c es

a t th e carb o x y term in u s o f Vmw IE 1 7 5 .

Table C5.6

A. COOING POTENTIAL OP V TEEMINUS OP Vmw IE 175 mKNA- - — r ^ — — ~ , - . . . - _ , ^

UGA s to p codon

v a l - s e r - l e u - g l y - l e u - a l a - h i s - g l y - p r o - g l n - a r g - h i s - a r g - a l a - g l y -

g l y - g l y - g l y - g l y - a r g - g l y - g l y - a l a - g l y - g l y - g l y - g l y - g l y - l e u - g l y -

h i s - a l a - p r o - a l a - a l a - g l y - s e r - c y s - g l y - l e u - g l y - a r g - a r g - l e u - g l y -

a r g - a r g - a r g - a r g - a r g - a r g - v a l - a r g - g ly - g ly - a r g - g ly - a la - v a l - t h r -

g ly -a r g -a s p -g ly -a la -g ly -a r g - le u -C O O H

UAA s to p codon

i l e - a r g - t r p - a l a - c e r - a l a - t h r - a l a - a r g - s e r - g l y - t h r - v a l - l e u - a l a -

a l a - a l a - g l y - a l a - v a l - g l u - v a l - l e u - g l y - a l a - g l u - a l a - g l y - l e u - a l a -

t h r - p r o - p r o - a r g - a r g - g lu - v a l - v a l~ a s p - t r p - g lu - g ly - a la - t r p - a s p -

g lu -a s p -a s p -g ly - g ly -a la - p h e -g lu - g ly -a s p -g ly - v a l - le u - C O O H

B. AMIBO ACIB USAGB

amino a c id UGA s to p codon UAA s to p codon

pro 2 2

a rg 15 4

a la 8 12

g !y 24 9

o th e r s 29 31

to bal 68 5B

( c o n t i n u e d o v e r l e a f )

T able C5»6 co n tin u ed

G. THItfD CODOLi POSITION USAGE

n u c le o t id e UGA sto p codon UAA sto p codon

G 2 4 1 3° 1' 44

1[52C

t .

oCM 22 J

A 4 ]' 25 ’ 8

T 12 j 4 j

Figure C3.1

D ata f o r S s t I r e s t r i c t i o n maps o f HSV-1 BamH I k and HSV-2 BamH I g .

A 1 HSV-1 ■BamH I k2 HSV-1 BamH I 33 HSV-1 BcoR I k

D ig e s ted[ w ith S s t I ; a g a ro se g e l .4

B 1 HSV-2 BamH I2 HSV-2 BaraH I u3 HSV-2 BcoR I k4 HSV-2 BcoR I ra!? HSV-2 Bamli I uvw6 HSV-2 BcoR I £-~

D ig e s te d . w ith S s t I ; 2 ‘» a g a r o se g e l .

C 1 HSV-2 BamH I £2 HSV-2 BamH I u3 HSV-2 BcoR I k4 HSV-2 BcoR I m

D ig e s ted w ith S s t I ; '7 • 5; * p o ly a c rylam:

D 1 i.iSV-2 Damn I u2 HSV-2 npn I a

D ig e s te u w ith S s t I ; 1 .3 > a g a r o se g e l

B 1 HSV-2 Damn I2 HSV-2 Kpn I r s

D ig e s te d WitxJ S s t I ; 2?* a g a r o se g e l .

Bach o f t lis aoove fr a g m en ts was prex>ared from n ic k t r a n s la t e d nSV-1 o r nSV—2 DBA.

S o t I fra g m en ts o f Damn I j o i n t or te r m in a l fra g m en ts a re i n d ic a t e d , A .d e n o te s a genome te r m in a l fra g m en t.

* I H H ! !CO

CN

Ol

m 1m cb

i ioo

t- CM CO ^ in co

Is-

CD

in

co

CN

^ _ i _____ i i 1 1^ It *•- » .'•-4

i x a t I9

I * 1

6-|

Fig u re 0 3 . 2

Some d a ta f o r Sma I r e s t r i c t i o n maps o f HSV-1 BamH I k.

1 HSV-1 BamH I k2 HSV-1 BamH I k3 IISV-1 BamH I g4 HSV-1 BamH I k5 HSV-1 BamH I k6 HSV-1 BamH I g Sau5A

I A d ig e s t e d w ith Sma I B d ig e s t e d w ith Sma I G d ig e s t e d w ith Sma

2fs> a g a ro se g e l .

B 1 pBR322 H in f :2 HSV-1 BamH I3 HSV-1 BamH I4 HSV-1 BamH I

■ 5 HSV-1 BamH I6 HSV-1 BamH I7 HSV-1 BamH I8 HSV-1 BamH I

S s tS s t

5/^ p o ly a c r y la m id e g e l .

C 1 pBB322 H inf I m arkers2 -6 IISV-1 BamH I k Sau3A I A d ig e s t e d w ith

in c r e a s in g am ounts o f Sma I

2 fa agaro s e g e1 .

[ m arkersk d ig e s t e d w ith Sma I 1 Sau3A I A d ig e s t e d w ith Smak Sau3A I B d ig e s t e d w ith Smak Sau3A I C d ig e s t e d w ith Sma

B,B d ig e s t e d w ith SmaA d ig e s t e d w ith Sma I C d ig e s t e d w ith Sma I

JJ 1 HSV-1 BamH I k S s t w ith Sma I~

I C p a r i i a l l y d ig e s t e d

5 ft p o ly a c r y la m id e g e l .

B 1 iiSV-1 BamH I m Sau3A I B p a r t i a l l y d ig e s t e d w ith Sma I

3; p o ly a c r y la m id e g e l .

Sma I r e s t r i c t i o n fra g m en ts o f HSV-1 BamH I k a re in d ic a te u in p a n e ls A and 3 . S i z e s o f p a r t i a l d i g e s t i o n p r o d u c ts in bp a re in d ic a te d in p a n e ls C-B w itii Sma I r e s t r i c t i o n fra g m en ts in l e t t e r s .H ick t r a n s la t e d p lasm id nil A was U3ea.Sma I fra g m en ts o f iiSV-1 BamHI x Sau3AI A (430 bp i sp a r t o f Smal A) and a 1631 bp maricer a re in d ic a t e d in p a n e l F,

§ g< l" " f *L L U .O

Ui|

□ I

CO

If)

CO

CM

rs. O) CO CO

CO

CO O o> CO CM CM

I 1

I II

I II

I IJ I M

’■ I t n I Iui o m o m o mu-

1 o ^ 2(O ^0)0) co ^

I Ih- O)O CO CM

1 II I f Ii i i ii r ~ i i i<C O O L U LU I )O o

1 I "T ~05 g COCM Is*

/

I I Io q- a

CO

m

CO

CM

1—

<1

I II If

M i t I 1 I I I •

i t t f i i n< DQ O LU U - I 0 — “ )

Q

Figure C3.5

R e s t r i c t i o n maps o f HSV-1 BamH I R (u pp er p a n e l)

and. HSV-2 BamH I g ( lo w er p g n e l ) , w hich c o n ta in

th e j o i n t o f L and S . The r e c t a n g le in d ic a t e s

th e £_>osition o f th e a se q u e n c e , and b oth fra g m en ts

a r e ab ou t 6000 bp in s i z e . .

N om en cla tu re has been a s s ig n e d to th e fr a g m en ts o f

some d i g e s t s . Sac I and S s t I a re is o s c h iz o r a e r s .

R e s t r i c t io n maps f o r genome t e r m in i, ea ch o f

w hich h as a te r m in a l a seq u e n c e , may r e a d i ly be

deduced from th o se o f th e j o i n t r e g io n s ( s e e

i l g u r e 0 3 .1 3 ) .

I- 1L_JSac 1 B T A t c ToSau3A 1 T A T B Tot c tTaq 1 A 1f f B T cHinc II tSma 1 t TT TT t TT T T T trr t

J C L D IMH 1 KPG A R F B MQE O

nUSac 1 4 T 3 2 Tat 1 T sSau3A 1 T T T TTaq 1 t T T T T THinc II t TKpn 1 rSma 1 TT TT3 116 7 4 ir

9i f r

2 1°125T1 8

figure C3.4

A u to ra d io g ra p h sh ow in g BamHI r e s t r i c t i o n p r o f i l e s o f

i n v iv o ^ P - l a b e l l e d v i r i o n DNA from te n s t o c k s o f

IiSV-1 s t r a i n 17 and t h e i r p a r e n t s to c k ( P ) , and from

te n s t o c k s o f HSV-2 s t r a in HG52 and t h e i r p a r e n t

s to c k ( P ) . Gel e l e c t r o p h o r e s i s was c a r r ie d o u t

on 0 .7 $ (HSV-1) and 1 $ a g a r o se (H SV -2). J o in t and

te r m in a l fr a g m e n ts , and fra g m en ts e x h ib i t in g s i z e

v a r i a b i l i t y , a r e i n d ic a t e d . R e s t r i c t i o n maps a re

shown in F ig u r e s C l018 and C l .1 9 .

i n 111 u i iii I Ii f *

IUIUIMIIII1 1 1 I ! 1 1

m iu a u u i ' ! 1 * f f4 4 • 1 4 • *

mitt n u n ! Ill* Imui nuii i UUl 1m m uni r im i n i i 1 till 11 it in man i! itnitiuatii i f t K f 1I I I I I I

m m n u n 1 Ii nut man ii i 1 ? I 1 ’ 1 I 1 1 I 1m m ua in i r tilt i

I || I 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1a -a>*- o v - ' ^ -r c x n CO UD 105 E w £ >

>

i i i i i _ i ro "o m h- o) .

o

u / m i m

i i i i i i n

m u inm i l h i

mmiH i n

m u inm u inm u i n

m«i:&m e i m

m e t w t

m « i i «T TTTi in it i 11 i » i i m a -v *-a ■ - -* —g c 0 ?OO O ) W w Q .

II 1 1 1 II M «- > jg K > N

F igu re C 3.5

LEFT HAND PANEL

Autorad&graph show ing Smal r e s t r i c t i o n p r o f i l e s o f

Bam HI j o in t fra g m en ts i s o l a t e d from i n v iv o ^ P -

l a b e l l e d LNA from te n HSV-1 s t r a in 17 s t o c k s , t h e ir

p a r e n t ( P ) , and a m ix tu re o f th e te n fra g m en ts (M).

Gel e le c t r o p h o r e s i s was c a r r ie d o u t on a 7 * 5 $

p o ly a c r y la m id e g e l , and th e p o r t io n below J

r e p r e s e n ts a lo n g e r ex p o su re o f th e g e l . Fragment

s i z e s (bp) were c a l ib r a t e d w ith H a e l l l and H in fl

d i g e s t s o f pBR322 DNA ( S u t c l i f f e , 1 9 7 8 ) , and s i z e

r a n g es o f th e v a r ia b le fra g m en ts Smal A and G a re

in d ic a t e d . The Smal map i s shown in F ig u re C 3 .3 .

KIGIIT I1ANP PANEL

A u torad iograp h show ing TagI and Sau3AI p r o f i l e s o f' ' ' 32BamHI j o in t fra g m en ts i s o l a t e d from in v iv o P -

l a b e l l e d DNA from f i v e o f th e ten HSV-1 s t r a in 17

s t o c k s , w ith t h e i r p a r e n t ( P ) . E le c t r o p h o r e s is was

c a r r ie d o u t on a 1 .5 # a g a r o se g e l . TagI fra g m en ts4 _

a re in d ic a t e d on th e l e f t and Sau3AI fra g m en ts on

th e r i g h t . The p o s i t i o n s o f m o le c u la r w e ig h t

m arkers o f 1631 and 516 bp a r e shown. Maps a re

g iv e n in F ig u re C3.3*

< CQ O

*tO I

_ 00 I

S " I3 T-(0

CO 0 .

05 | |

to I ICO I

CM § |

« *“ ^I - 0 .

I I I In CD CJ £<o K

o

2 O O o O n O o o2 CO *fr£ ooIs- mm ^ to cm cmi i i ii i i i i -1

50”

143

t « I i t « %G ) 1 II 1 1 ! i0 0 • H 1 1 II I t •

1 II 1 111 I I iCO 1 II 1 1 1 ii o “ M l 1 1 1 1 l l

1 II 1CO u t i i i i t •CM i i i i i i i i ir - i i i i i i i

i i i i i i i i f0 - ' i i i i /

i ) i i ii i i i i VflOO IU U_ I -----5

Q g 111111111 jii_j

r - <£> 00 COO) CO Tt TtI I I I

I I

I I

I I I IO Q -O CC

<3

-38

F igu re C3»6

LEFT HAND PANEL

A u to ra d io g ra p h show in g Smal r e s t r i c t i o n p r o f i l e s o f32

BamHI j o i n t fr a g m en ts i s o l a t e d from i n v i v o P -

l a b e l l e d LNA from te n HSV-2 s t r a in HG52 s t o c k s , t h e ir

p a r e n t ( P ) , and a m ix tu r e o f th e te n fra g m en ts (M ).

E le c t r o p h o r e s is was c a r r ie d o u t on a 7.57*

p o ly a c r y la m id e g e l , and th e p o r t io n below 6

r e p r e s e n t s a lo n g e r ex p o su re o f th e g e l . Fragment

s i z e s a r e g iv e n in T ab le C 3 * l, and th e Smal map in

F ig u re C 3 .3 .

EIGHT HAND PANEL

Smal / -BamHI r e s t r i c t i o n p r o f i l e s o f recom b in an t

p la sm id s b ea r in g th e BamHI j o in t fra g m en t o f HSV-1

s t r a i n 17 or HSV-2 s t r a i n HG52 LNA* 2 j*g LNA was

a p p lie d to ea ch t r a c k o f a p o ly a c r y la m id e g e l ,4

w h ich a f t e r e l e c t r o p h o r e s i s was s ta in e d w ith e th id iu m

brom ide and p h otograp h ed under u l t r a v i o l e t i l lu m in a t io n .

The p la sm id v e c t o r pATl53 c o n ta in s no Smal s i t e s and

th e r e fo r e m ig r a te s a s a la r g e fragm en t in ea ch t r a c k .

C lo n es k l , k2 and k3 c o n ta in th e HSV-1 j o i n t fragm en t

w ith a s in g l e a se q u e n c e , x l and §2 c o n ta in th e same

fragm en t w ith two a se q u e n c e s , and g l c o n ta in s th e

nSV-2 j o i n t fragm en t w ith two a seq u en ces* HSV-1

Smal fra g m en ts a r e in d ic a t e d on th e l e f t , w ith s i z e

r a n g e s o f A and G, and s i z e s i n bp o f 0X174 Hael l l

m arxers on th e r i g h t . S tandard r e f e r e n c e num bers o f

th e c lo n e s a re g iv e n in Table B 1 .3 -

M k1

k2 k3

k1 k2

gl M

f2£?C M tO O ’- ’- St ^ 00 _ .o T-oor^ to 01 ’- £

S 2 2 o o tO COCMCM N r 7I I I I I I I I ' 1

I------- w

3

J

- -"T*

v _ »

7 1

i n

u<

I I II I I I I H, 1 1COO^ L*- X 3 ~} ^ Z

Q U Jo

\

i i i ieg coTt in

iCO

I I I I IIs- co o o n

Homology betw een th e j o in t r e g io n s o f th e

genomes o f HSV-1 and HSV—2 .

Recom binant p la sm id s c o n ta in in g th e HSV-1 or HSV-2

3amH I j o in t fra g m en ts were d ig e s t e d and tr a n s fe r r e d

to n i t r o c e l l u l o s e from a 4 $ p o ly a c r y la m id e g e l .

B lo t s t r i p s were th en h y b r id is e d w ith n ic k

t r a n s la t e d recom b in ant p la sm id DIJA.

1 k l Barnh I/Sm a I h y b r id is e d w ith k l

2 k l Barnh I/Sm a I n y o r id is e d w ith g l

3 . k l Bamil I / Sma I / S s t I h y b r id is e d w ith k l

4 k l Bamh I / Sma I / S s t I h y b r id is e d w ith g l

3 g l Bam a I / Sma I n y O rid ised w ith g l

6 g l Barnh i / Sma I n y O rid ised w ith k l

hSV-1 Barnh I k Sma I fra g m en ts are in d ic a t e d f o r

tr a c k s 1 - 4 , tn o se fra g m en ts in p a r e n th e s e s i n d ic a t in g

th e new p o s i t i o n s o f m ig r a tio n o f p o r t io n s o f Sma I

fra g m en ts a f t e r c le a v a g e w ith S s t I . HSV-2

Bamii I g Sma I fra g m en ts a re in d ic a te d f o r t r a c e s 3 - 6 .

Smal maps a r e snown in f ig u r e s C 3.3 and C 3 .1 3 .

fragm ent J o f g l , s in c e t h i s p lasm id h as two a

se q u e n c e s , maps in a l o c a t io n sp a n n in g th e a -a

ju n c t io n ( f ig u r e C 3 .1 3 ) .

3 4

G = *

(E)l J -

Some d a ta f o r the r e s t r ie t iu n m apping o f th e 1760 bp H in f I fragm en t w hich c o n t a in s th e j o i n t betw een HSV-1 L a n d S.-

A 1 Sau3A I A d ig e s t e d w ith H in f I2 Sau3A I A d ig e s t e d w ith HTnf I/Sm a I3 Sau3A I A d ig e s t e d w ith Sma I .

B *1 BamH I k d ig e s t e d w ith Sma I2 S /U d ig e s t e d w ith Sma I3 Sau3A I A d ig e s te d ~ w ith Sma I4 §7H d ig e s t e d w ith Ava I5 S/H d ig e s t e d w ith Ava I I6 S/H d ig e s t e d w itn Ava I l / Sma I7 S /n d ig e s t e d w ith BstN I8 S/II d ig e s t e d w ith BstN i/S m a I9 S/H d ig e s t e d w ith S ae I I

10 S /ii d ig e s t e d w ith Hae I I / Sma IM pBH322 Hae I I I m arkers ~*

C M pBR322 Hae I I I m arkers1 S/H d ig e s t e d w ith Sma I2 S /ii d ig e s t e d w ith S s t I I / Sma I3 S/H d ig e s t e d w ith S s t I I4 S/H d ig e s t e d w ith BgT I / Sma I5 s / i i d ig e s t e d w ith B gl I

A l l th e fra g m en ts were p rep ared from n ic k t r a n s la t e d p la sm id Li\lA c o n ta in in g HSV-1 BamH I k ( k l in P ig u re 0 3 . S ) . ,S/H r e f e r s to th e Sau3A I /H lh f I 1130 bp fra g m en t w hich c o n ta in s th e j o in t betw een L and S i n th e HSV-1 genom e. 7*b<<> p o ly a c r y la m id e g e l s were u se d .In p a n e l A the Sma I fra g m en ts o f Sau3A I A are in d ic a t e d 7 in c lu d in g th e 430 bp fra g m en t wKich i s partv o f Sma I A. The Sma I fra g m en ts o f BamH I k a r e in d ic a t e d in p a n e l and th e s i z e s o f pBB322r llae I I I m arkers in bp in p a n e ls B and C.

:::

i i i00 co coin CM

9 I H I I II

T—in

5 m i M i 1£ V . iO) 3 m

CO X )

CO X ; i iin S t

■ Bco H E f l H B ■ i i 3cm 2)

i i i i «%«

I I ;CQ < “ 0 ^ 0 -

i n io q- a

CM

Icc

Figure C5«9

R e s t r ic t io n e n d o n u c lea se maps o f th e j o i n t r e g io n s

o f HSV-1 s t r a in 17 and HSV-2 s t r a in HG52, and th e

n u c le o t id e seq u e n c in g s t r a t e g i e s em ployed . The

HSV-1 fragm en t (H in fI 1760 bp) c o n t a in s th r e e

r e i t e r a t i o n s shown in b la c k ( I , I I , I I I ) . R e s t r ic t io n

s i t e s in t h i s r e g io n o f th e DHA o f HSV-1 s t r a i n

USA-H were i d e n t i c a l to th o s e in s t r a in 1 7 ,

e x c e p t f o r an e x t r a Hinf I s i t e i n th e c seq u en ce

50 bp from th e a se q u e n c e . The HSV-2 fragm en t i s

S s t I 1310 bp ( BamHI g S s t I 2 ) .

L o c a t io n s o f th e d i r e c t r e p e a t s a t th e b -a and a - c

ju n c t io n s a re in d ic a t e d .

...........b ; .......... a ' 4 ............. C '* 4 .........

^ .............................. ►

i I I i

Hin

f I

Sst

II Ss

t II

BstN

1

Has

II Sm

a \J

Bg

l I

Bgl

1 Ss

t II

Sma

\J

Sma

1./

Sst

II Av

a II

Sst

IISm

a l.i

Ava

1 Ha

e II

Sau3

A

Taq

1M

bo

II Ha

e II

Taq

1 B

sfN

I Ha

e II

Sst

II

if!

iHi

nf

1

1 W l l n W ______________ I I I V O

->i n m

8-

*i

oo-

CMo- « f o a f o 1000 1200 1400 1 6 0 0 1766 bp

M 2 «C - Z ^** 3t +* +* 5 c fi cM j (0 (A > « ^ 3 2o o to m < to z < z

w i i n w w i

(0 X » I Z ( A ( 0 <0 <0 <

1200 1310 bp800 1000

F igure 0 3 . IQ

F in er d e f i n i t i o n o f the lo c a t io n o f HSV-1 a sequence

v a r i a o i l i t y .

Sau3A I A was i s o l a t e d from n ic k t r a n s la t e d p la sm id s

k l (1) and k 2 ( 2 ) , which c o n ta in i n s e r t s o f

hSV-1 BamH I k, and the l a t t e r o f which has a

l a r g e r Sma I (J fragment (F igure 03# 6 ) .

The two Sau3A I A fragm ents were c le a v e d as

i n d ic a t e d , and in eacn case the fragm ent which i s

la r g e r in k2 i s shown. The pBH322 Hae I I I marker

fragm ents (h) are in c lu d e d .

F igure C 5.l l

N u c le o t id e seq u en ces o f th e j o i n t r e g io n s o f HSV-1 and

HSV-2 Dl\iA, shown c o n v e n t io n a l ly 5 ' - 3 ' in the b ' - a ' - c 1

o r i e n t a t i o n .

A HSV-1 s t r a i n 17 c lo n e h i , w ith the p o s i t i o n s where

HSV-1 s t r a i n USA-8 d i f f e r s in d ic a t e d below the sequ en ce .

A dash r e p r e s e n t s the absen ce o f a n u c l e o t id e , and s t r a in

USA-8 was n o t sequenced between th e two s e t s o f th ree

d o t s . The l o c a t i o n s o f the genomic ter m in i w ith r e s p e c t

to th e j o i n t seq u en ces are shown ( s t r a i n 17 above and

s t r a i n USA-8 below the s e q u e n c e ) . These d e f in e th e

b -a and a -c ju n c t io n s and are c l o s e to the r i g h t hand

ends o f d i r e c t r e p e a t s (21 n u c l e o t id e s in s t r a i n 1 7 ,

17 in s t r a i n USA-8), the common 17 n u c l e o t id e s o f which

are boxed (311 -32 7 and 7 1 0 - 7 2 6 ) . R e i t e r a t io n s I , I I

and I I I are i n d ic a t e d , and the two AATAAA h e x a n u c le o t id e s

are boxed (1500 -1 505 and 13 4 6 -1 5 4 1 )• The r e g io n in

wnich the te r m in a l coded n u c le o t id e a t the 3' term inust ^

o f Vmw ih 175 mHHA was mapped i s shown (1530-1525)*

a number o f r ep ea ted s t r u c t u r e s o f unknown s i g n i f i c a n c eit

are p r e se n t but are n o t d i s c u s s e d in th e t e x t ( e . g . an* t

a lm o st e x a c t 18 n u c l e o t id e in v e r te d r e p e a t a t 243-260

and 2 8 1 -2 9 2 and a d i r e c t r e p e a t o f 20 n u c le o t id e s in

s t r a i n U3a- 8 te r m in a t in g a t 778 and 8 6 1 ) . The e n t i r e

sequence lias a GO c o n te n t o f 7 8 N.

H HSV-2 s t r a i n nGt>2. Tne l o c a t i o n s o f th e genomic

term in i w ith r e s p e c t to tn e j o i n t sequence are shown.

Tney are c l o s e to the r ig h t nand ends o f 17 n u c le o t id e

d i r e c t r e p e a t s ^ooxed) ana d e f in e tne p -a ana a -c

j u n c t io n s . The e n t i r e sequence has a GC c o n te n t o f 80?*.

GACTCGGGAA CGTGGAGCCA CTGGCGCAGC AGCAGCGAAC AAGAAGGCGG GGGCCCACCG GCGGGGGGCG 701 A

GCGGCGGGGC GGCCGCGGGC GCGCTCCTGA CCGCGGGTTC CGAGTTGGGC GTGGAGGTTA CCTGGGACTG 140

TGCGGTTGGG ACGGCGCCCG TGGGCCCGGG CGGCCGGGGG CGGCGGGGGC CGCGATGGCG GCGGCGGCGG 210

GCCATGGAGA GAGAGAGCGT GCCGGGGTGG TAGAGTTTGA CAGGCAAGCA TGTGCGTGCA GAGGCGAGTA

b'/la'GTGCTTGCCT GTCTAACTCG CTAGTCTCGG .CCGCGGGGGG CCCGGGqT-GC CCGCCGCCAC CGCTTTAAAG

G-

GGCCGCGCGC GACCCCCGGG GGGTGTGTTT TGGGGGGGGC CCGTTTTCGG GGTCTGG C C T

reiteration I( CCGCTCCTCCCC)18 CCGCTCC CGCGGCCCCG CCCCCCACGC(CCGCTCCTCCC—) g ( CCGTCTGTGGGTGGGGCTCCTCCC) g

280

350

407

650

CCGCCGCGCG CGCGCACGCC GCCCGGACCG CCGCCCGCCT TTTTTGCGCG CGCGCGCGqT'cGCGGGGGGC I G CGC C

S m al a f \ °GCGGGCJGCC ACAGGTGAAA----------- CCAACAG-----------------AGC ACGGCGCACT CCGCACGT-----------------------CA

C T CAACA AACACCAAC C GGCG TCCAGTTCCTCATCVCACGTCACGT CATCCACCAC ACCTGCCCAA CAACACAACT CACAGCGACA ACTCACCGCG CAACAACTCC

720

770

840

TGTTCCTCAT CCACACGTCA CCGCGCACCT CCCGCTCCTC CAGACGTACC CCGGCGCAAC ACACCGCTCCC A C G

reiteration IITGCTACACAC CACCGCC ( CCCTCCCCAGCCCCAG) 2 ( CCCTCCCCGGCCCCAG) g CCCTC

TC CCAG ( CCCTCCCCAGCCCCAG)15+ . . .

910

1060

CCCGGCGCGT CCCGCGCTCC CTCGGGGGGG TTCGGGCATC TCTACCTCAG TGCCGCCAAT CTCAGGTCAG 1130

reiteration IIIAGATCCAAAC CCTCCGGGGG CGCCCGCGCA CCACCA (CCGCCCCTCGCCCCCTC)19 C CGCCCCTCC^

V,. IE 175 mRNA 3 '-end_ _ _ _ 4 ...................................ATAA^CAACG CTACTGCAAA ACTTAATCAG* GTTGTTGCCG [TTTATTGCGT CTTCGGGTCT CACAAGCGCC

1500

1570

CCGCCCCGTC CCGGCCCGTT ACAGCACCCC GTCCCCCTCG AACGCGCCGC CGTCGTCTTC QTCCCAGGCG 1640

CCTTCCCAGT CCACAACTTC CCGCCGCGGG GGCGTGGCCA AGCCCGCCTC CGCCCCCAGC ACCTCCACGG 1710

CCCCCGCCGC CGCCAGCACG GTGCCGCTGC GGCCCGTGGC CGAGGCCCAG CGAATC 1766

Bb'A a'

TGGCGTGCGC AGCCCGGGCC GTGTTGCGGG CCCTCTTAAG GGGCGGCGGC AGGACGGGGA CTpCCGCCCC 70

GCCTCTTTTC CCCCGGGGAG TCAACCCCCG GGGGGGGTGT TTTTTGGGGG GGGGCGCGAA GGCGGGCGGC 140

GGCGGCGGGC GGGCGGCAGG GCAGCCCCGC GCGCCCCCTT CCCCGTCCCT CCCCCGGAGC CGGCCGCTCC 210

CCCGCGGGCG CCGCCCCTCC CCCCGCGCGC CGCGGGCTGC CTTCCGCGGC GCCCCCGCGC G.G

a > — ^ — iC'280

CCGCGCCCGC CCCCGCgCGC^jAGGACGGGG^CJAGCAGGC TGTGCCGCAG ACCACCACAC ACTCCCAA 348

Figure C3.12

A u to ra d io g ra p h show ing s i z e s o f genom ic te r m in a l

r e s t r i c t i o n fra g m en ts o f HSV-1 s t r a in s 17 and USA-8.3220 /*g HSV-1 UNA was P - l a b e l l e d a t 5 1 te r m in i i n a

volum e o f 2 0 y u l, and th e BamHI S and L te r m in a l

fra g m en ts i s o l a t e d . These w ere r e c le a v e d a s

in d ic a t e d and e l e c t r o p h o r e s i s c a r r ie d ou t on a

1 6 # p o ly a c r y la m id e (8 M u r e a ) se q u e n c in g g e l , u s in g

a s s i z e m arkers G-A and C-T c h e m ica l d e g r a d a tio n

n u c le o t id e se q u e n c in g r e a c t io n s (k in d ly p ro v id ed

by M -J. M u rch ie ), th e fra g m en t s i z e s o f w hich a r e

g iv e n in n u c l e o t i d e s . The S te r m in a l BamHI fragm en t

was co n ta m in a ted w ith th e L te r m in a l fragm en t

p o s s e s s in g an a d d i t io n a l a se q u e n c e .

5 I

I I

25

20

Figure C3.13

H e s t r ic t io n maps o f th e BamHI fr a g m en ts o f HSV-1

UNA (u p p er p a n e l) and HSV-2 UNA ( lo w e r p a n e l)

w hich c o n ta in th e j o in t (6 kbp) and te r m in a l

r e g io n s . The two te r m in i a re a l ig n e d w ith th e

j o i n t so th a t th e p o l a r i t i e s o f th e th r e e r e g io n s ,

in c lu d in g th e a se q u e n c es ( r e c t a n g l e s ) , a re

e q u iv a le n t . The rem ainder o f th e genome i s

in d ic a t e d by a d o tte d l i n e . The S s t I map i s

shown above th e genom e, and th e Smal map b elow .

and kg d en o te S s t I and Smal te r m in a l fra g m en ts

o f th e genom e.

The p r e se n c e o f two tandem d i r e c t l y r e p e a te d a

se q u e n c es a t th e HSV-1 j o i n t , a s shown a t th e

bottom o f th e upper p a n e l , in tr o d u c e s a secon d

Smal G fra g m en t, and a fragm en t P 1 o f 43 bp w hich

i s th e same s i z e a t Smal P . The p r e se n c e o f two

tandem d i r e c t l y r e p e a te d a seq u e n c es a t th e HSV-2

j o i n t , a s snown a t th e bo i/fcom o f th e lo w er p a n e l ,

in tr o d u c e s a secon d Smal 12 fra g m en t, and a

fragm en t J o f 2^0 bp.

B C D

JOINTI | | II I III I

J C L D NH I KPGI I III I R F B M Q E O

TERMINUS

B X L

I II II I III I J C L D NH I KPGXl

TERMINUS

C I D

I I I IXSG A RF B MQE O

JOINT with 2 a sequences

B C D I I

I II II I III II I I I III IJ C L D NH I KPGP'G A R F B MQE O

BJOINT

4 3 7I II

II II II 3 1167 4 9

8

IB I 2 10125

5 6

84 I 3 n X L

TERMINUS

TERMINUS

JOINT with 2 a sequences

II II II 3 1167 4 9

i7;

n M M3 1167 4 9

III2 K)I2Xl

Xs 8-* II □ — I I

X sl25

c u ll I I

2 10 J 5 12 12

5 1 6

8

I 5 . 6

I8

X

gigure Q3.14

LEPT HAND TWO PANELS

A u to ra d io g ra p h o f 8 $ p o ly a c r y la m id e se q u e n c in g g e l s

show in g r e i t e r a t i o n I o f HSV-1 s t r a i n s 17 and USA-8.

Prom l e f t to r ig h t th e fragm en t seq u en ce shown i n ea ch group i s a s f o l l o w s .

1 . S t r a in 17 (p la sm id k l ) from Aval 370 to Avai l 67 0 .

2 . S t r a in 17 (p la sm id k l ) from Avai l 670 to B stN I 1 3 0 .

3 . S t r a in 17 (p la sm id k2) from Avai l 670 to BstN I 1 3 0 .

4 . S tr a in USA-8 from A v a il 670 to Aval 3 7 0 .

The r e i t e r a t e d seq u en ce i s shown i n ea ch c a s e , and t h e i r cop y number in p la sm id s k l and k 2 .

RIGHT HAND TWO PANELS

A u to ra d io g ra p h o f 6 ‘/Z p o ly a c r y la m id e se q u e n c in g g e l s4

show in g r e i t e r a t i o n I I o f HSV-1 s t r a in 17*

Prom l e f t to r ig h t th e fragm en t seq u en ce in ea ch group i s a s fo l lo w s *

1 . S t r a in 17 (p la sm id k l ) from S s t l l 710 to Sau3AI 1 1 3 0 .

2 . S t r a in 17 (p la sm id k2) from Avai l 670 to Sau3AI 1 1 3 0 .

Copy numbers o f th e two r e p e a te d u n i t s , w hich d i f f e r

by a s i n g l e n u c le o t id e , a re shown by l e t t e r s and num bers.

The main r e i t e r a t e d seq u en ce i s n o te d .

The tr a c k s a re in grou p s o f f i v e a s f o l l o w s .

G - c le a v e d a t G r e s id u e sA - c le a v e d a t G and A r e s id u e sAC - c le a v e d a t A and, to a l e s s e r e x t e n t , C r e s id u e s T - c le a v e d a t C and T r e s id u e sC - c le a v e d a t C r e s id u e s .

The r e s t r i c t i o n s i t e s and n u c le o t id e p o s i t i o n s ( to th e n e a r e s t 10 bp) r e fe x r e d to a r e shown in P ig u re C3*9.

oI-

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F igure 0 3 .1 5

LEFT HAND TWO PANELS

A u to ra d io g ra p h o f 8/» p o ly a c r y la m id e se q u e n c in g g e l s

sh ow in g r e i t e r a t i o n I I I o f HSV-1 s t r a in 1 7 .

From l e f t to r ig h t th e fra g m en t seq u en ce i n each group i s a s f o l l o w s .

1 . S t r a in 17 (p la sm id k l ) from TagI 1500 to Sau3AI 1 1 3 0 .

2 . S t r a in 17 (p la sm id k l ) from Sau3AI 1130 to TagI 1 5 0 0 .

3 . S tr a in 17 (p la sm id k2) from Sau3AI 1130 to TagI 1 5 0 0 .

The r e i t e r a t e d seq u en ce i s n o te d , and th e copy numbersin p la sm id s k l and k 2 .

RIGHT HAND THREE PANELS

A u to ra d io g ra p h o f 6 $ p o ly a c r y la m id e se q u e n c in g g e l s

show ing th e d i r e c t r e p e a t s a t th e a - c and a - a ju n c t io n s

o f HSV-1 s t r a i n 17 and a t th e c - a ju n c t io n o f HSV-1

s t r a i n USA-8.

From l e f t t o r ig h t th e fragm en t seq u en ce i n ea ch group i s a s f o l l o w s .

1 . S t r a in 17 (p la sm id k l ) from Avai l 670 to Sau3AI 1 1 3 0 .

2 . S t r a in 17 (p la sm id £2 ) from Avai l 670 to M nll 4 2 0 . T h is p la sm id h as two a s e q u e n c e s , so th e n u c le o t id e seq u en ce p ro ce ed s from th e Avai l s i t e i n one a se q u e n c e , a c r o s s th e ju n c t io n betw een th e tw o“ a s e q u e n c e s , to th e Mnll s i t e in th e n e x t .

3 . S t r a in USA-8 fro m -H in fI 770 to H a e ll 1 6 0 .

The r e p e a te d seq u en ce i s n o te d ; t h a t in (3 ) i s from th e com plem entary s tr a n d o f th e d u p lex to t h a t shown in F ig u r e 0 3 .1 1 * from r ig h t to l e f t . The lo c a t io n s o f M nll s i t e s were deduced from F ig u re 03*11• S t r a in USA-8“h a s an a d d i t io n a l H in fI s i t e a t 770 compared w ith s t r a i n 1 7 .

Track d e s ig n a t io n i s th e same a s f o r F ig u re 0 3 * 1 4 .The r e s t r i c t i o n s i t e s and n u c le o t id e p o s i t i o n s( to th e n e a r e s t 10 bp) r e f e r r e d to a re shown in F ig u re 0 3 .9 .

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o o o o o0 0 , 0 0 0 rfO o o o o

Figure 03.16

L o c a t io n o f th e 3' term in u s o f HSV-1 Vmw IE 175

mRNA w ith r e s p e c t to th e DNA se q u e n c e .

The Taq.I D fragm en t c o n s i s t in g o f n u c le o t id e s

1 4 9 3 -1 6 0 8 was l a b e l l e d a t th e 3 ! te r m in i and s e p a r a te

s tr a n d s w ere i s o l a t e d . The s tr a n d com plem entary to

Vmw IE 175 mRNA ( t h a t shown i n F ig u r e 0 3 *11) was

i -y b f id is e d to RNA a s d e sc r ib e d i n th e t e x t , t r e a te d w ith

SI n u c le a s e and e le c tr o p h o r e s e d a lo n g s id e th e DNA seq u en ce

o f th e same fragm en t ( 3 f - 5 f r e a d in g from th e bottom ) on

an 8 fo p o ly a c r y la m id e (8 M u re a ) g e l . The tr a c k s a r e a s

f o l l o w s .

1 - u n tr e a te d s in g l e s tra n d ed DNA fragm en t

2 - a n n ea led w ith 10 fAg y e a s t RNA ( c o n t r o l )

3 - a n n ea led w ith 5 j jg II3V-1 p o ly a d e n y la te d IE mRNA

4 - a n n ea led w ith 10 iISV-1 p o ly a d e n y la te d IE mRNA

5 - a n n ea led w itn 15 HSV-1 p o ly a d e n y la te d IE mRNA

6 - a n n ea led w ith i s o l a t e d 4 .7 kb IE mRNA w hich c o d e s f o r

T racks G, A, T and C in d ic a t e th e DNA se q u e n c in g r e a c t io n s

a s d e s ig n a te d in F ig u re 0 3 .1 4 .

1 2 3 4 5 G A T C 6

S t r u c tu r a l arran gem en ts a t th e j o in t and te r m in i

o f th e HSV genome (a b o v e ) , and a t j o i n t s c o n ta in in g two

a seq u e n c e s ( b e lo w ) . The a se q u e n c e s a r e shown a3

r e c t a n g le s and t h e i r o r ie n t a t io n and th e l o c a t i o n s . o f

th e d i r e c t r e p e a t s a t th e b -a and a - c ju n c t io n s a re

i n d i c a t e d .

SECTION 4

INVERSION OP THE TWO SEGMENTS

OP THE HSV GENOME

- 130 -

RESULTS

In tr o d u c t io n

doth o r ie n t a t io n s o f th e L and S segm en ts o f HSV-1 and

113V-2 v ir io n DNA a re p r e s e n t i n eq u a l am ounts, g iv in g r i s e to

fo u r genome arra n g em en ts . T h is i s a l s o a f e a t u r e o f HSV-l/HSV-2

i n t e r t y p ic reco m b in a n ts . The mechanism by w hich segm ent

in v e r s io n o c cu r s i s unknown. V ir io n DNA o f th e recom b in ant

B x l^ 2 8 ), a r e s u l t o f g e n e t ic r e c o m b in a tio n betw een HSV-1 tsB

and IISV-2 t s l , was shown by P r e s to n e t a l . (1 9 7 8 ) to be a t y p ic a l

in p o s s e s s in g 1 p red o m in a n tly in one o r i e n t a t io n , w hereas

S was p r e s e n t e q u a lly in b oth o r i e n t a t io n s (P ig u r e C 4 .3 ) .

T h is p henotype was d e f in e d a s Mf ix e d " , norm al in v e r s io n b e in g

termed " fr e e " . I t m ust be s t r e s s e d t h a t i n f ix e d reco m b in a n ts

th e m a jo r ity , but n o t 'the e n t i r e t y , o f v i r i o n DNA m o le c u le s

p o s s e s s th e a p p r o p r ia te segm ent i n th e m ajor o r i e n t a t io n .

P r e s to n e t a l . (1978) co n clu d ed from g r o s s r e s t r i c t i o n

a n a ly s i s th a t B x l(2 8 ) p o s s e s s e s HSV-2 seq u en ce in TR and HSV-1

seq u en ce in IR^. One p o s s i b i l i t y , t h e r e f o r e , i s t h a t a r e g io n

o f t y p e - s p e c i f i c hom ology betw een TR and IRj, i s n e c e s s a r y f o r

normal in v e r s io n o f 1 . However, o th e r reco m b in a n ts a ls o

seemed h e t e r o ty p ic f o r TR j/lR^ and y e t in v e r t e d n o r m a lly .

I t i s n o t p o s s ib le by g r o s s r e s t r i c t i o n a n a l y s i s to d i s t in g u i s h

betw een HSV-1 and HSV-2 DNA seq u e n c es in r e g io n s c l o s e r to th e

j o in t or te rm in i th a n 4000 bp, so in o r d er to l o c a t e p r e v io u s ly

u n d e te c te d c r o s s o v e r s th e j o i n t and te r m in a l r e g io n s were

a n a ly sed in d e t a i l f o r B x l ( 2 8 ) , f o r B x l(2 8 ) s u b c lo n e s , and

fo r a number o f o th e r reco m b in a n ts .

The recom b in an t KH4 was g e n e r a te d by marker r e sc u e o f

HSV-1 t s B DNA by HSV-2 DNA fra g m en ts (W ilk ie e t a l . , 1 9 7 9 b ) .

KH/| was a n a ly se d in d e t a i l in th e work d e s c r ib e d h ere and

shown to oe f ix e u in L and S, a l lo w in g in v e r s io n o f both

- 131 -

segm en ts to be i n v e s t i g a t e d .

The g e n e s c o d in g f o r V ^ IE 110 (H SV -l) and i t s c o u n te r p a r t

in II3V-2, Vfi.w IE 1 1 8 , map e n t i r e l y w ith in TR and IHj,. P r e s to n

e t a l . (1 9 7 8 ) showed t h a t reco m b in a n ts w M ch 'a re h e t e r o t y p ic

f o r t h i s d ip lo id gene in d u ced b oth IE 110 and IE 1 1 8 . Two

s u b c lo n e s o f B x l(2 8 ) a re shown h ere to be d e le t e d in TRj, or IRj,,

and th e s e a llo w e d i n v e s t i g a t i o n o f th e e x p r e s s io n o f t h i s gene"

by genomes w ith o n ly one f u l l co p y .

In th e f o l lo w in g d e s c r ip t io n s i t i s ap p aren t t h a t th e

i n t e r p r e t a t io n o f d e t a i l e d r e s t r i c t i o n a n a ly s e s o f recom b in ant

genome s t r u c t u r e s i s v er y com p lex . T h e r e fo r e , a lth o u g h a

s i g n i f i c a n t p r o p o r t io n o f d a ta h a s been in c lu d e d i n th e

P ig u r e o , i t s in t e r j j r e t a t io n h as been l im i t e d to a few exam p les.

Emphases a r e p la c e d on s t r u c t u r e s c r i t i c a l t o th e c o n c lu s io n s

drawn, on d e m o n str a tin g th e l i m i t s o f th e a n a l y s i s , and on

show ing th e s t r u c t u r a l s u b t l e t i e s o f w h ich HSV recom b in an t

genom es a re c a p a b le .

S tr u c tu r e s o f th e .jo in ts and te r m in i o f B x l ( 2 8 ~ l )- • ^ • - -

The w ork ing s to c k u sed in t h e s e ex p er im e n ts was named

B x l ( 2 8 - 1 ) , th e term B x l(2 8 ) b e in g r e t a in e d f o r th e p u ta t iv e

o r i g i n a l recom b in an t a s d e s c r ib e d b e lo w . The DNAs o f t s B , t s l

and B x l (2 8 -1 ) were ^2P - la b e l l e d by n ic k t r a n s l a t io n and c le a v e d

w ith S s t I o r Smal and th e p r o d u c ts se p a r a te d by e le c t r o p h o r e s i s

on 2 $ a g a r o s e , 7 .5 $ p o ly a c r y la m id e o r 1 5 $ p o ly a c r y la m id e g e l s .

The r e s u l t i n g p r o f i l e s were a n a ly se d u s in g th e r e le v a n t

r e s t r i c t i o n maps o f th e s e r e g io n s o f th e genome (P ig u r e C3#3)*

C lea v a g e p r o d u c ts o f te r m in i w hich do n o t co rresp o n d to

fra g m en ts from th e j o in t r e g io n , and a re th e r e fo r e th e

te r m in a l Ss t I or Smal fra g m en ts o f th e genom e, a re r e f e r r e d to

a s X ( s e e P ig u re C 3 .1 3 ) . I t was som etim es n e c e s s a r y to c le a v e

c o n ig r a t in g fra g m en ts ( e . g . HSV-2 BamHI v w ), and in t h e s e

- 132 -J

in s t a n c e s a t t e n t io n i s drawn o n ly to c le a v a g e p r o d u c ts from /ir

j o in t or te r m in a l r e g io n s . P a in t bands a r i s i n g from

c o n ta m in a tin g se q u e n c e s , a fe a tu r e o f th e u se o f n ic k

t r a n s la t e d DUA, a re a l s o n o t d is c u s s e d .

F ig u re C 4.1 p a n e l A shows th e S s t I c le a v a g e p r o f i l e s o f

B x l(2 8 -1 ) j o i n t fr a g m e n ts . Track 2 shows th e m ajor j o in t

fra g m en t, w h ich i s ty p e 1 , and a ls o HSV-2 S s t I fra g m en ts from

th e b and c seq u e n c es i n l e s s e r abundance. The la r g e r j o in t

fragm en t ( tr a c k 3 ) h as bands c h a r a c t e r i s t i c o f both th e HSV-1

and IISV-2 j o in t r e g io n s , and p rob ab ly c o m p r ise s m a in ly th e ty p e

1 j o in t w ith an e x tr a a seq u en ce (HSV-1 S s t I A i s la r g e r ) and

ty p e 1 IHl jo in e d to ty p e 2 IRg ( th e n o v e l j o i n t fragm en t

p o s s ib ly m ig ra ted above IISV-2 S s t I 1 , p erh a p s w ith two a

s e q u e n c e s ) . Two r a r e r form s may a l s o be p r e s e n t : one w ith

ty p e 2 IR^ jo in e d to ty p e 1 IRg, and th e o th e r w ith HSV-2 DNA

'throughout th e j o i n t . These r e s u l t s show t h a t th e m ajor j o i n t

s p e c ie s i s ty p e 1 th ro u g h o u t, but t h a t o th e r s p e c ie s due to

in v e r s io n a r e a l s o p r e s e n t . P a n el B tr a c k 2 shows a Smal

d i g e s t o f th e m ajor j o i n t o f B x l ( 2 8 - 1 ) , in w hich a g a in th e

m ajor bands a re ty p e 1 . HSV-1 Smal G, b e in g a fragm en t v a r ia b le

in s i z e , i s s m a lle r in t h i s recom binant th a t in t s B . Track 3

o f p a n e l C and tr a c k 3 of* p a n e l D show t h a t th e m ajor L

term in u s i s ty p e 2 , in c lu d in g th e a se q u e n c e . The HSV-1 m inor

L term in u s i s b a r e ly d e t e c ta b le in p a n e l C tr a c k 4 , s in c e L

i s f i x e d .

P r e s to n e t a l . (1 9 7 8 ) d em on strated t h a t th e HSV-2 Kpnl

s i t e in TRg/IRg was to some e x te n t s e g r e g a te d betw een b oth

TRg and IRg in B x l ( 2 8 - 1 ) , and so th e two S te r m in i were

i s o l a t e d s e p a r a t e ly . The un ique HcoRI S term in u s o f B x l ( 2 8 - 1 ) ,

c o n v e n t io n a l ly th e r ig h t hand s id e o f S , c o n ta in s HSV-1 and

- 133 -

HSV-2 S s t I fragm en ts c o n s i s t e n t w ith i t b e in g a m ix tu re o f

th e norm al IISV-2 S te r m in a l BcoRI fra g m en t and a fragm en t o f

s im ila r s i z e w ith a c r o s s o v e r in TRg (F ig u r e C 4.2 p a n e l A;

summarised in F igu re C 4 .1 8 ) . S m all s i z e d i f f e r e n c e s betw een

some fra g m en ts from p a r e n ta l and recom b in an t genom es ( e . g .

HSV-2 S s t I 1) a re p ro b a b ly th e r e s u l t o f s i z e v a r i a b i l i t y in

th e s e r e g io n s . The S s t I p r o f i l e o f th e B g ll l S te r m in u s ,

c o n v e n t io n a l ly th e l e f t hand s id e o f S , i s c o n s i s t e n t w ith i t

b e in g a m ixtu re o f th e norm al HSV-2 S te r m in a l B g ll l fragm en t

and a fragm en t w ith a c r o s s o v e r i n TRg in an e q u iv a le n t

l o c a t io n to th a t d e s c r ib e d above (F ig u r e C 4.2 p a n e l C ).

P a n e ls B and 1) o f F ig u r e C4 .2 d em o n stra te th e p r e se n c e o f th e

Kpnl s i t e i n HSV-2 TR3/IK 2 in a t l e a s t a p r o p o r t io n o f both

S term in a l fra g m en ts of- B x l ( 2 8 - l ) . Smal d ig e s t io n o f th e

BamHI S te r m in a l fra g m en ts o f B x l ( 2 8 - l ) (F ig u r e C 4.2 p a n e l E)

shows th e p re sen c e o f an HSV-1 a seq u en ce a t th e ty p e 1

term in u s and an HSV-2 a seq u en ce a t th e ty p e 2 te rm in u s ,

deduced in t h i s c a s e from th e p r e se n c e o f HSV-2 Smal £ .

These r e s u l t s co n firm th a t B x l ( 2 8 - l ) i s f i x e d in L and

show t h a t , in th e m ajor o r ie n t a t io n o f 1 , TR and IR-^,

in c lu d in g txie a se q u e n c e s , a re h e t e r o t y p ic . "S c o n s i s t s o f a

m ixtu re o f a t l e a s t two s t r u c t u r e s , th e c r o s s o v e r i n one o f

w hich i s p rob ab ly m ost a c c u r a t e ly lo c a t e d w ith in HSV-1 BamHI m! ,

b ecau se su b c lo n e s o f B x l(2 8 -1 ) w hich p o s s e s s HSV-1 BamHI q

la c i: t h i s fragm en t (F ig u r e C 4 . l l p a n e l C ). A summary o f th e

s tr u c t u r e s p r e s e n t in B x l ( 2 8 - l ) , and t h a t o f th e p u t a t iv e

o r ig in a l recom b in ant B x l ( 2 8 ) , a re i l l u s t r a t e d in F ig u re 0 4 .1 8 .

G ross s t r u c t u r e s o f B x l ( 2 8 - l ) s u b c lo n e 3

TWenty su b c lo n e s o f 8 x 1 (2 8 -1 ) were i s o l a t e d by Mrs

m. Murphy by th re e c y c le s o f p laq u e p u r i f i c a t i o n a t 31°>

- 134 -

and in v iv o ^2P - la b e l l e d DNA was a n a ly se d u s in g X b a l, Hindl l l ,

BcoRI, B g l l l , Hpal , Kpnl and BamH I. R e s u lt s f o r s e v e r a l o f th e

s u b c lo n e s w ith fo u r e n d o n u c le a se s a re shown in F ig u r e s C4«4 and

0 4 .3 . Genome s t r u c t u r e s deduced from t h i s g r o s s a n a ly s i s a re

shown in F ig u re 0 4 .6 .

More th an h a l f o f th e s u b c lo n e s a re f r e e i n L, and a l l

a re f r e e i n S . Those su b c lo n e s f ix e d i n L a re r e a d i ly

i d e n t i f i e d , a s i s B x l ( 2 8 - l ) , by th e overabundance o f th e two

j o in t fr a g m en ts HSY-2 EcoRI c and e o v e r th e o th e r tw o, b and

d, and by th e e x c e s s o f th e L te r m in a l fra g m en t HSV-2 Hpal g

o v er HSV-1 Hpal m (F ig u r e C 4 .4 ) . The o r i g i n a l c r o s s o v e r in

i s c o n ser v e d in a l l s u b c lo n e s , and a l l e x c e p t perhaps

su b c lo n e 27 have sym m etric S seg m en ts: t h a t i s , e i t h e r S

c o n s i s t s e n t i r e l y o f HSV-2 DNA se q u e n c e , o r i t h a s HSV-1 DNA•»

a t both S te r m in i w ith c r o s s o v e r s w ith in th e two HSV-1 BamHI m1

fra g m en ts (F ig u r e 0 4 .1 1 p a n e l C ). The d a ta s u g g e s t t h a t two

su b c lo n e s a re d e le t e d i n T R j/lR ^; su b c lo n a 14 w ith 3 x 10^

d a l to n s d e le t e d in ty p e 1 su b c lo n e 22 w itn 5 x 10

d a lto n s d e le t e d i n ty p e 2 IR^. R e s t r i c t i o n p r o f i l e s o f

th e s e two s u b c lo n e s show n o v e l bands in t e r p r e t e d a s j o in t and

te r m in a l fra g m en ts r e s u l t i n g from th e d e l e t i o n s . Only su b c lo n e

27 has i d e n t i c a l r e s t r i c t i o n p a t t e r n s to B x l ( 2 8 - 1 ) .

The a n a ly s i s a l s o s u g g e s t s t h a t some f r e e su b c lo n e s such

a s 1 0 , 11 and p erh ap s 9 have a d d it io n a l c r o s s o v e r s in TR o r

IRl . T h ere fo re th e j o in t and te r m in a l fra g m en ts o f c e r t a in

s u b c lo n e s were a n a ly se d in d e t a i l by c le a v a g e w ith S s t I and Smal .

S tr u c tu re s o f th e .jo in ts and te r m in i o f B x l(2 8 -1 ) s u b c lo n e s

F ig u r e s 0 4 .7 and C 4.0 show d a ta f o r j o in t r e g io n s ,

F ig u r e s C 4.3 and C 4.10 f o r 1 t e r m in i , and F ig u r e s C 4 . l l and

C 4.12 f o r 3 t e r m in i. To a la r g e e x t e n t o n ly th e m ajor genome

- 135 -

form s w ere a n a ly sed in 'these e x p e r im e n ts .

The m ajor j o in t o f su b c lo h e 1 , w hich i s f ix e d in L, i s

ty p e 1 th rou ghou t (F ig u r e C 4.7 p a n e l A tr a c k 3 , p a n e l C tr a c k

3 , F ig u re C 4.8 p a n e l A tr a c k 2 ) , in c lu d in g th e a seq u en ce from

w hich HSV-1 Smal G com igrated w ith Smal I . The m ajor L

term in u s i s typ e 2 , in c lu d in g th e a seq u en ce (F ig u r e 04*9

p a n e l A tr a c k 2 , F ig u re 0 4 .1 0 p a n e l A tr a c k 6 j . The m inor

ty p e 1 L term in u s i s b a r e ly d e t e c t a b le i n th e S s t I p r o f i l e

(F ig u r e 0 4 .9 p a n e l A tr a c k 3 ) , a s e x p e c te d s in c e L i s f i x e d .

The S term in u s i s typ e 1 (F ig u r e C 4«H p a n e l A tr a c k 3 ,

F ig u re 0 4 .1 2 p a n e l A tr a c k 5 ) . T h ere fo re su b c lo n e i s

h e t e r o ty p ic f o r TR and IRj,, in c lu d in g th e a s e q u e n c e s , w hereas

TRg and IRg a re id e n t i c a l ."

S u b clon e 5 i s a ls o f ix e d i n L and s im i la r i n s tr u c tu r e to

su b c lo n e 1 e x c e p t th a t th e c r o s s o v e r s i n TRg/lRg a r e i n HSV-1

Bamli l q Smal F r a th e r th an in HSV-1 BamHI mf » ( e .g . F ig u re

C 4.7 p a n e l A tr a c k 4 , F ig u re 0 4 .1 2 p a n e l A tr a c k 6 ) .

S u b clon e 9 i 3 f r e e i n L and h a s a more com plex s t r u c t u r e .

HSV-2 BamHI v , th e norm al ty p e 2 L te rm in u s , was n o t d e te c te d

in th e g r o s s a n a ly s i s (F ig u r e 0 4 .5 ) , s u g g e s t in g th a t b oth L

te r m in i a re typ e 1 a s in d ic a te d i n F ig u re 0 4 * 6 . In d ee d , t h i s

wan su p p orted by th e f in d in g t h a t th e m ajor j o in t o f norm al

s i z e i s type 1 th ro u g h o u t (F ig u re 0 4 .7 p a n e l A tr a c k 5 , p a n e li—>

C tr a c k 4 , F ig u re 0 4 .8 i^anel A tr a c k 3)> and t h a t th e L

term in u s w hich com igrated w ith HSV-1 BamHI s i s ty p e 1

(F ig u r e C 4.9 i)an el A tr a c k 6 , F ig u re 0 4 .10 . p a n e l A tr a c k 2 ) .

I t i s n o ted th a t HSV-1 Smal P i s a few bp s m a lle r i n t h i s

su b c lo n e than in tsB . Bands r e p r e s e n t a t iv e o f th e ty p e 2

L term in u s v/ere d e te c te d i n th e S s t I d i g e s t o f th e S term in u s

(F ig u r e 0 4 .1 1 p a n e l A tr a c k 5 ) . T h e r e fo r e , a lth o u g h th e

norm al typ e 2 L term in u s i s n o t p r e s e n t , t h i s seq u en ce i s

- 136 -

r e p r e s e n te d in a fragm en t w hich m ig ra ted a t a p o s i t i o n

0 .3 x 10^ d a lto n s l a r g e r . In o rd er t o c l a r i f y t h e s e r e s u l t s ,

th e L te r m in i were i s o l a t e d s e p a r a t e ly v i a th e u n iq u e Hpal

fra g m en ts and c le a v e d w ith S s t I (F ig u r e C 4.9 p a n e l B t r a c k s

3 and 4)» The BamHI L term in u s from HSV-1 Hpal ift i s ty p e 1

a s e x p e c te d , but t h a t from HSV-2 Hpal g c o n s i s t s o f a m ix tu re

o f two s p e c i e s . The f i r s t i s a norm al ty p e 1 BamHI s fra g m en t,

and th e secon d i s a ty p e 2 term in u s v /ith an a d d i t io n a l 0 .3 x 10

d a lto n s o f DNA a t th e te r m in u s . In d e e d , HSV-2 Hpal g o f

su b c lo n e 9 c o n s i s t s o f a fragm en t o f norm al s i z e and one a

l i t t l e la r g e r (F ig u r e 0 4 * 5 ) . The p r e c i s e n a tu r e o f th e la r g e r

fragm en t was n o t d e term in ed , but i t seem s from Smal a n a ly s i s

n o t to te r m in a te i n a ty p e 2 a s e q u e n c e . The m ost l i k e l y

e x p la n a t io n o f th e s e r e s u l t s i s t h a t su b c lo n e 9 c o n s i s t s o f a

m ix tu re o f two s t r u c t u r e s d i f f e r i n g a t one L te r m in u s , a s

i n d i c a t e d in F ig u re C 4 .1 7 . The ty p e 1 a seq u en ce i s a l i k e l y

c a n d id a te f o r th e a d d i t io n a l DNA a t th e ty p e 2 term in u s o f

su b c lo n e 9b , b ut t h i s was n o t p ro v en . The a c t u a l e x p la n a t io n

may be .more com p lex , but i t i s c l e a r t h a t f u r t h e r re c o m b in a tio n

h as o ccu rred a t th e 1 te r m in i o f th e su b c lo n e 9 genom e,

g e n e r a t in g r e g io n s o f t y p e - s p e c i f i c hom ology betw een TR and IR^.

S u b clon e 29 h as 'two* ty p e s o f L t e r m in i . One i s th e

norm al ty p e 2 term in u s and th e o th e r c o n t a in s HSV-1 S s t I B

but a s m a lle r te r m in a l fragm en t S s t I X (F ig u r e C 4.9 p a n e l A

tr a c k s 7 - 9 , F ig u r e C 4.10 p a n e l A tr a c k 3 J . T h is i s c o n s i s t e n t

w ith th e o b s e r v a t io n o f HSV-2 BamHI y and a s l i g h t l y s m a lle r

fragm en t in th e g r o s s BamHI p r o f i l e (F ig u r e C 4 .5 ) . F ig u re

C 4.10 p a n e l A tr a c k 4 shows t h a t th e s m a lle r term in u s c o n s i s t s

o f HSV-1 DNA e x c e p t t h a t th e HSV-1 a seq u en ce h as been

r e p la c e d by an HSV-2 a seq u en ce ( s e e a l s o F ig u re C 4.9 p a n e l

13 tr a c k s 3 -6 and F ig u r e C 4.13 p a n e l D t r a c k s 3 - 4 ) . ^ne gmai i e r

- 137 -

s i z e i s e x p la in e d by th e f a c t th a t th e HSV-2 a seq u en ce i s

a p p r o x im a te ly lpO bp s m a lle r than th a t o f HSV***l ( s e e S e c t io n 3 ) .

The c r o s s o v e r i s lo c a t e d in HSV-1 Smal P , w hich sp an s th e

b -a j u n c t io n , g e n e r a t in g a new fragm en t o f 28±2 bp a s shown

i n F ig u re 0 4 .1 8 . Both th e norm al ty p e 2 term in u s and th e

n o v e l one a r e r e f l e c t e d i n p r o f i l e s o f j o in t fra g m en ts

(F ig u r e C4*7 p a n e l A tr a c k 6 , p a n e l C t r a c k 5 , F ig u r e C4*8

p a n e l B t r a c k s 2 - 3 ) . The S term in u s i s ty p e 2 (F ig u r e C 4 .H

p a n e l A tr a c k 6 , F ig u re 0 4 .1 2 p a n e l A tr a c k 8 ) . S u b clon e 2 9 ,

w hich i s f r e e i n 1 , has t y p e - s p e c i f i c hom ology betw een

TRl and IR^ w h ich i s co n fin e d e s s e n t i a l l y to th e ty p e g a

se q u e n c e s .

F ig u re C 4.8 p a n e l C shows t h a t th e j o in t r e g io n o f

su b c lo n e 14 h a s HSV-1 a and c se q u e n c e s , but fr a g m e n ts ,

t o t a l l i n g o n ly 600 bp o f HSV-1 Hpal m a d ja c e n t to th e Hpal

s i t e i n th e b seq u en c e . I n te r v e n in g b seq u en ce fr a g m en ts a r e

a b s e n t . These r e s u l t s con firm th a t 3 x 10 d a lto n s o f DNA

h ave been d e le t e d from IR- a d ja c e n t to th e a s e q u e n c e . The

predom inant L term in u s i s " t y p e 2 (F ig u r e 04*9 p a n e l C t r a c k 3>

F ig u re C 4.10 p a n e l B tr a c k 4 ) , and th e S ter m in u s i s ty p e 1

(F ig u r e C 4 . l l p a n e l B t r a c k 4 , F ig u re C 4.12 p a n e l B t r a c k 4 ) .

S u b clon e 22 has a Hpal L term in u s o f a p p r o x im a te ly

1 .4 x 10^ d a lto n s (2400 b p ), w hich i s ab ou t 5 x 10^ d a lto n s

s m a lle r than th e e q u iv a le n t fragm ent in t s l . C lea v a g e o f

t h i s fragm en t w ith Smal produced fo u r d e te c te d fra g m en ts o f

1 0 0 0 , 8 5 0 , 300 and 55 bp. The o rd er o f t h e s e fra g m e n ts from

th e L term in u s i s 3 0 0 -1 0 0 0 -5 5 -8 5 0 bp, and was deduced a s f o l l o w s .

The 300 bp fragm en t co rresp o n d s to HSV-1 Smal G from th e norm al

HSV-1 1 term in u s o f su b c lo n e 22 (F ig u r e 0 4 .1 0 p a n e l C );

th e r e fo r e th e n o v e l term in u s has a 'type 1 and n o t a ty p e 2 a

se q u e n c e . The 850 bp fragm ent i s s im i la r in , s i z e to th e o n ly

- 138 -

Smal fragm en t from IISV-2 Hpal g w hich was n o t produced from

HSV-2 Hpal f (F ig u r e 0 4 .1 0 p a n e l D t r a c k s 5 - 6 ) , and t h e r e fo r e

sp an s th e TR^-U^ J u n c t io n . I t i s n o t e x a c t ly th e same s i z e

b ecau se TR^-U^ ju n c t io n fra g m en ts a re somewhat v a r ia b le in s i z e

i n in t e r t y p ic 'r e c o m b in a n t s . The 55 bp fra g m en t i s common to

HSV-1 Hpal f and g and t h e r e f o r e i s c o n ta in e d w ith in TRj/ I R j .

The 1000 bp fragm en t m ust span th e d e le t e d r e g io n . I t i s ~

co n c lu d ed t h a t su b c lo n e 22 c o n ta in s no more than 1 .2 x 10

d a lto n s o f HSV-2 TR^, th e d e l e t io n e x te n d in g o v e r a p p ro x im a te ly

5 x 1 0 6 d a lto n s w ith in TR^. H y b r id is a t io n d a ta shown in

F ig u re 0 4 .1 2 co n firm ed t h e “ i d e n t i t y o f n o v e l J o in t and

te r m in a l fra g m en ts in r e s t r i c t i o n p r o f i l e s o f su b c lo n e s 14 and

2 2 , and d em on stra ted t h e ir freedom from c o n ta m in a tio n by

n o n -d e le te d genom es.

S tr u c tu r e s o f th e . jo in t s and te r m in i o f o th e r reco m b in a n ts

P r e v io u s ly d eterm in ed genome s t r u c t u r e s a r e shown in

F ig u re 0 4 .3 o f s e v e r a l reco m b in a n ts w h ich a p p a r e n t ly a re

h e t e r o ty p ic f o r a l l or p a r t o f TRL and IR- QTlTRg a n d ,IR g ,

and w hich y e t in v e r t n o r m a lly . T hese w ere a n a ly se d f o r

u n d e te c te d c r o s s o v e r s .

The BamHI p r o f i l e o f B x l ( 3 l - 2 ) i s shown in F ig u re 0 4 * 1 4 .

The J o in t fra g m en t i s la r g e r th a n t h o s e o f th e p a r e n ta l v i r u s e s ,

and when c le a v e d w ith S s t I produced HSV-2 IRj, and HSV-1 IRg

fra g m en ts (F ig u r e 04*13 p a n e l A t r a c k 2 ) . The ty p e 2 1— ~ 6 term in u s c o r r e sp o n d in g to t h i s J o in t fragm en t i s 0*3 x 10

d a lto n s la r g e r th an HSV-2 BamHI v , and d id n o t produce th e

u s u a l ty p e 2 te r m in a l Smal X fragm en t (F ig u r e 0 4 .1 3 p a n e l A

tr a c k 3 , p a n e l B tr a c k 3 ) . The o th e r L term in u s ( t r a c k 5)

i s ty p e 1 , w ith a la r g e r Smal G fragm en t th a n tsB w hich i s

a l s o p r e s e n t a t th e ty p e 2 1 term in u s and a t th e J o in t

(p a n e l n tra ck s 2 , ; j , 5 ) . Tuese r e s u l t s s u g g e s t th a t one

- 139 -

L term in u s o f B x l(3 1 -2 ) i s ty p e 1 , and th a t th e o th e r i s ty p e 2

w ith th e a d d it io n o f a ty p e 1 a seq u en ce a t th e term in u s

a d ja c e n t to a ty p e 2 a se q u e n c e . I s o l a t i o n o f th e two L

te r m in i s e p a r a te ly and d ig e s t io n w ith S s t I showed a norm al

IISV-1 1 term in u s (F ig u r e 0 4 .1 3 p a n e l C tr a c k 4 ) , and a fragm en t

c o n t a in in g , among c o n ta m in a tin g fr a g m e n ts , HSV-2 S s t I 2, &&& i

hut a la r g e r S s t I te r m in a l fragm en t ( t r a c k 3 ) . I s o l a t i o n o f

th e two S s t I L te r m in a l fra g m en ts and d i g e s t i o n w ith Smal

showed th e norm al HSV-1 term in u s and one c o n s i s t in g o f th e

IISV-2 term in u s w ith th e a d d it io n o f an HSV-1 a seq u en ce

a d ja c e n t to th e HSV-2 a seq u en ce a t th e term in u s (F ig u r e 04*13

p a n e l D t r a c k s 3 - 4 ) . The J o in t fragm en t c o r r e sp o n d in g to th e

norm al ty p e 1 L term in u s was n o t a n a ly s e d , so i t i s shown w ith

a s in g l e a seq u en ce in F ig u r e C 4.17 a lth o u g h i t p ro b a b ly

c o n ta in s more than o n e , in v iew o f th e a b se n c e o f HSV-1 BamHI k

o f norm al s i z e i n F ig u re 04*14 p a n e l A# I t i s co n c lu d ed from

th e s e r e s u l t s th a t B x l ( 3 1 - 2 ) , w hich in v e r t s n o r m a lly ,, h a s

t y p o - s p e c i f i c hom ology betw een HSV-1 a se q u e n c es a t th e J o in t

and 1 te r m in u s .

3 x 6 ( 1 7 - 6 ) , RS6 and £ 1 3 -2 were a n a ly se d s im i l a r l y , and

d a ta a rc shown in F ig u r e s 04*14 and 04*15* The r e s t r i c t i o n s i t e

betw een HSV-2 S s t I 2 ajad i s a b se n t i n B x 6 (1 7 -6 ) , b u t S s t I 2

i s p r e s e n t . The c r o s s o v e r in IRj, o f R 13-2 i s lo c a t e d w ith in

HSV-1 Smal N.

D x l(2 ) was i n i t i a l l y th o u g h t to be h e t e r o t y p ic f o r

TKj/IR^ and T R g/lR g. The BamHI J o in t fragm en t i s 0 .3 x 10^

d a lto n s la r g e r than t h a t o f HSV-1 o r HSV-2 DNA, and Smal

d i g e s t i o n snows i t to c o n s i s t o f HSV-1 IR^ seq u e n c es a d ja c e n t

to HSV-2 IRg se q u e n c es w ith one in te r v e n in g a seq u en ce o f each

ty p e (F ig u r e 0 4 .1 6 p a n e l A tr a c k 3 , F ig u r e s 0 4 .1 7 and C 4 I1 8 ).

in o x e io r e a sequcx.ce hom ology betw een J o in t and te r m in i i s

- 140 -

m a in ta in ed f o r both L and S . A s im ila r s t r u c t u r e w ith th e a

se q u e n c e s r e v e r se d in o rd er was i d e n t i f i e d i n a su b c lo n e o f

su b c lo n e 2 o f B x l ( 2 8 - l ) (d e sc r ib e d below ; F ig u re C 4.16 p a n e l

A tr a c k 4 ) , and i s a ls o i l l u s t r a t e d in F ig u r e C 4*18. The b a s ic

s tr u c t u r e o f D x l(2 ) shown in F ig u re C4*17 i s f u r th e r c o m p lica te d

by th e p r e se n c e o f a p r o p o r tio n o f L te r m in i w ith an a d d i t io n a l

ty p e 2 a seq u en ce a t th e term in u s (F ig u r e 0 4 * 1 6 p a n e l C tr a c k

3 , p a n e l I) t r a c k 3 ) . T h is i s even fu r th e r c o m p lic a te d by

s e g r e g a t io n o f th e se q u e n c e s in TR g/lR g. For in s t a n c e i n F ig u r e

04*16 p a n e l B HSV-1 S s t I C and £ and HSV-2 S s t I £ and 6 a re

p r e s e n t in TRg and IRg, w hich were i s o l a t e d s e p a r a t e ly .

These f e a t u r e s o f th e L and S te r m in i a r e a l s o ap p a ren t in th e

d a ta o f F ig u re 04*13 p a n e l H tr a c k s 7 -1 0 , b u t have n o t been

in c lu d e d in F ig u re 04*17.

E x p r e ss io n o f IE 1 1 0 /1 1 8 by B x l(2 8 -1 ) s u b c lo n e s

To i n v e s t i g a t e th e e x p r e s s io n o f IB 1 1 0 /1 1 8 by su b c lo n e s

14 and 2 2 , th e genomes of. w hich a re d e le t e d in TR^ o r Ifi^ ,

in f e c t e d c e l l x^ olypeptid es were prepared from t h e s e , and”

o th e r reco m b in a n ts , and su b je c te d to p o ly a c ry la m id e g e l

e le c t r o p h o r e s i s by Hr II .S . Marsden and Mr G-. Hope.

Im m ediate e a r ly p o ly p e p t id e s were ind u ced i n HFL c e l l s

(a l i n e o f human f o e t a l lu n g c e l l s e s t a b l i s h e d by Hr B. C a r r i t t

in th e I n s t i t u t e o f G e n e t ic s , G lasgow) a s d e s c r ib e d by

P r e s to n e t a l , (1 9 7 8 ) . The recom b in an ts u se d produced

e q u iv a le n t p laq u e numbers on b oth BHK 013 and HFI c e l l s .

The a e in o d in v o lv e d i n f e c t i o n o f c e l l s a t h ig h m u l t i p l i c i t y

and in c u o a t io n fo r 3 hr a t 37° in th e p r e s e n c e o f

c y c lo h e x im id e . The drug was removed and p o ly p e p t id e s l a b e l l e dd 9w ith ^ b -m etn ion in e in th e p resen ce o f a c t in o m y c in H. Whole

- 141 -in f e c t e d c e l l p o ly p e p t id e s were p rep ared by l y s i s i n s i t u

o f th e c e l l s (M arsden e t a l . , 1 9 7 8 ) . S in c e IE 110 h as been

shown to p a r t i t i o n w ith th e n u c le u s ( P e r e ir a e t a l . , 1 9 7 7 ) , in

some e x p er im e n ts n u c le i were p rep ared by tre a tm e n t o f c e l l s

w ith 0 .6 5 $ NP40 in i c e - c o l d i s o t o n i c b u f f e r , b e fo r e b o i l in g4

i n d e n a tu r in g b u f fe r .

F ig u r e C 4 .2 0 , w hich i s a co m p o site o f S H S -p o lyacry lam id e

g e l s from s e v e r a l e x p e r im e n ts , show s im m ed iate e a r ly

p o ly p e p t id e s in d u ced by HSV-1 s t r a in 1 7 , HSV-2 s t r a i n HG52

$nd s e v e r a l reco m b in a n ts , th e genome s t r u c t u r e s o f w hich are

shown in F ig u re 0 4 .1 9 . L anes 1 -1 0 show w hole in f e c t e d c e l l '

p o ly p e p t id e s , and la n e s 1 1 -2 1 show n u c le a r p o ly p e p t id e s .

S ix HSV-1 coded (1 7 5 , 1 3 6 ' ( 1 4 3 ) , H O , 8 7 , 6 8 , 63) and s ix

HSV-2 coded (182~ 1 3 8 , 1 3 4 -1 3 2 , 1 1 8 , 6 7 , 6 4 ) IE p o ly p e p t id e s

can be i d e n t i f i e d i n t h e s e e x p e r im e n ts .

As o b serv ed p r e v io u s ly (P r e s to n e t a l . , 1 9 7 8 ) ,

reco m b in a n ts B x l(3 1 -2 ) and B x l ( 2 8 - 1 ) , w h ich a r e h e t e r o ty p ic f o r

th e r e p e a t s bounding UL, in d u ced b oth IE 110 (HSV-1) and th e

e q u iv a le n t HSV-2 p o ly p e p t id e IE 1 1 8 . S im i la r ly , su b c lo n e s 1

and 2 9 , w h ich a re h e t e r o ty p ic f o r TR and IR^, in d u ced both

IE 110 and IE 1 1 8 . The l a t t e r i n v e r t s n o rm a lly in L and S

b u t su b c lo n e 1 i s f ix e d , in L, and t h e r e fo r e w hether o r n o t th e

L segm ent in v e r t s n o rm a lly seem s n o t to a f f e c t e x p r e s s io n o f

th e IE 1 1 0 /1 1 8 g e n e s . S u b clon e 1 4 , w h ich la c k s HSV-1 seq u en ce

from IRT ( in c lu d in g a r e g io n from w hich th e 5 1 end o f th e mRNA

f o r IE 110 i s tr a n s c r ib e d ; F .J . R ixon , p e r s o n a l com m u n ica tion ),

in d u ced o n ly th e HSV-2 IE 1 1 8 . C o n v e r se ly , su b c lo n e 2 2 , w hich

la c k s HSV-2 seq u en ce from TR ( in c lu d in g th e w hole o f th e gene

f o r IE 118; A .J . E a sto n , p e r s o n a l co m m u n ica tio n ), in d u ced o n ly

th e HSV-1 IE 1 1 0 . S u b clon e 11 in d u ced IE 118 and a p o ly p e p t id e

w hich m igrate^ f a s t e r than IE 1 1 0 . F ig u r e 04*19 shows th a t a

c r o s s o v e r i s lo c a te d , w ith in th e DNA se q u e n c es c o d in g f o r th e

HSV-1 IE 110 icRNA. The a l t e r e d p o ly p e p t id e p ro b a b ly a r o se a s a

co n seq u en ce o f th e c r o s s o v e r p o s i t i o n i n IRj,. The a p p a r e n tly

s m a lle r s i z e may r e s u l t from n o n - c o l in e a r i t y o f i n i t i a t i o n or

te r m in a t io n cod on s f o r th e two polypeptides, a d e l e t io n o f some

c o d in g seq u en ce a s a r e s u l t o f th e c r o s s o v e r , o r from a l t e r e d

p r o c e s s in g o f th e h y b r id p o ly p e p t id e .

S t r u c tu r e s o f s u b c lo n e s o f B x l(2 8 -1 ) s u b c lo n e s

S ev en tee n su b c lo n e s each were i s o l a t e d by th r e e c y c l e s o f

p la q u e p u r i f i c a t i o n a t 31° from B x l(2 8 -1 ) s u b c lo n e s 2 and 29-,

in o rd er to d e term in e w h eth er a f ix e d su b c lo n e would g iv e r i s e

to f r e e p ro g en y , and w h eth er a f r e e su b c lo n e would g iv e r i s e

to f ix e d p ro g en y . The g r o s s s t r u c t u r e s o f th e progen y su b c lo n e s

were a n a ly se d u s in g Xbal , Hind l l l , EcoRI , B g ll l , Hpal , Kpnl and

BamH I, and a r e shown in F ig u re 0 4*21 .

S tr u c tu r e o f th e genome o f RE4

The r e s t r i c t i o n s i t e s in RE4 DNA were a n a ly se d i n th e

u s u a l way. H ind l l l and BamHI d i g e s t s a re shown i n F ig u r e

C 4.22 in t r a c k s l a b e l l e d R, The r e s u l t s o f t h e s e d i g e s t s and

o th e r s su g g e s te d t h a t th e KE4 genome i s d e le t e d in IR^ and th a t

a seq u en ce from e lse w h e r e h a s been in s e r t e d in p la c e Of IR^*

A l i k e l y e x p la n a t io n was th a t th e in s e r t e d DNA c o n t a in s

r e s t r i c t i o n s i t e s f o r H in d III and BamHI . th u s g e n e r a t in g

n o v e l fra g m en ts from one L term in u s ( i . e . th e f a i n t band

s l i g h t l y la r g e r th an HSV-2 H in d iII o , and th e band in te r m e d ia te

in s i z e betw een HSV-1 BamHI t and u v ) . The fragm en t s l i g h t l y

s m a lle r than HSV-1 H in d l l l o and th e fra g m en t in te r m e d ia te in

s i z e oetw een HSV-1 BamHI p and r may c o n ta in th e J u n ctio n

betw een norm al seq u en ce and the in s e r t e d seq u en ce

(fra g m en t T J ) . T hese fra g m en ts co u ld n o t be e x p la in e d a s

- 143 -r e s u l t i n g from norm al c r o s s o v e r s i n th e RE4 genome, and

n e i t h e r co u ld o th e r n o v e l fragm en ts th o u g h t to be J o in t

fra g m en ts r e s u l t i n g from th e n o v e l 1 te r m in u s , such a s th e

fragm en t w hich i s s l i g h t l y sm a lle r th an HSV-1 -BamHI k .

The p rop osed genome s tr u c tu r e o f RE4 i s i l l u s t r a t e d in

F ig u r e 0 4 .2 6 , and ex p erim en ts are d e sc r ib e d below i n su p p ort

o f t h i s u n u su a l arrangem ent.

The r e s u l t s o f ex p er im en ts in v o lv in g h y b r id is a t io n o f

HSV-2 H in d l l l n and o to n i t r o c e l l u l o s e s h e e t s c o n ta in in g

HSV-1, HSV-2 and RE4 Hindl l l o r BamHI d i g e s t s a r e shown in

F ig u re 0 4 .2 3 . HSV-2 Hind l l l n h y b r id is e d n o t o n ly a s

e x p e c te d to HSV-1 H in d l l l a p r e s e n t i n RE4 DNA, but a l s o to

th e n o v e l J o in t and te r m in a l fr a g m e n ts . HSV-2 H in d in o

h y b r id is e d i n each c a se to fragm en t TJ, th o u g h t to r e p r e s e n t

th e J u n ctio n betw een norm al and in s e r t e d s e q u e n c e s . The

i s o l a t e d RE4 H ind l l l TJ fragm en t h y b r id is e d to HSV-1

Hind l l l ab ed and BamHI b and f 1, and to HSV-2 H ind l l l b and o

and BamHI f , i and r . The h y b r id is a t io n r e s u l t s c e r t a i n l y

ca n n o t be e x p la in e d in term s o f l e g i t im a t e c r o s s o v e r s , b ecau se

RE4 H ind l l l TJ c o n ta in s seq u en ces from HSV-2 H in d l l l o

(HSV-2 BamHI f ) and IISV-2 H in d iII b (HSV-2 BamHI i r ) , w hich a re

n o t c o n t ig u o u s in th e HSV-2 genome. T hese e x p e r im e n ts

su p p ort th e h y p o th e s is th a t RE4 DNA i s d e le t e d in a t l e a s t

th e m a jo r ity o f IR^, w ith th e in s e r t io n o f an HSV-2 seq u en ce

from a s shown i n F ig u re 0 4 .2 6 .

To l o c a t e more p r e c i s e ly th e J u n ctio n betw een norm al and

in s e r t e d DNA th e BamHI and BamH I/H in d III TJ fra g m en ts were

i s o l a t e d and c le a v e d w ith a number o f r e s t r i c t i o n e n d o n u c le a se s

(F ig u r e 0 4 .2 4 ) . The r e s u l t s show t h a t Hind l l l TJ c o n ta in s

a lm o s t a l l o f HSV-2 Hind l l l o , w ith th e e x c e p t io n o f n o t more

than 25 bp a t the r ig h t hand end ( s e e Hhal d i g e s t s ) . R e s u lt s

w ith Hhal and A lu l show th a t n o t more than 260 bp o f HSV-2

- 144 -

BamHI i a re a d ja c e n t to HSV-2 H in d l l l o s e q u e n c e s . The r e s u l t s

o f t h i s exp erim en t have been in c lu d e d in F ig u r e C 4 .2 6 .

C lea v a g e o f th e RE4 BamHI n o v e l j o in t fragm en t

d em o n stra tes th e p r e se n c e o f HSV-2 IRg fra g m en ts b u t th e

a b sen c e o f IR- fra g m en ts o f e i t h e r HSV-1 o r HSV-2 (F ig u r e C 4.25

p a n e l A t r a c k '2 , p a n e l C t r a c k 2 , p a n e l D tr a c k 2 ) . I n s t e a d ,

Smal fra g m en ts c h a r a c t e r i s t i c o f HSV-2 BamHI r w ere produced

(p a n e l C t r a c k 2 ) . HSV-2 Smal £ aM 12 p r e s e n t , s u g g e s t in g

a ty p e 2 a seq u en ce a t t h e " j o in t ; th e a b sen ce o f HSV-2 Smal 10

shows t h a t n o t more th an a p p ro x im a te ly 45 bp o f th e HSV-2

b seq u en ce a r e a d ja c e n t to th e a se q u e n c e . These r e s u l t s and

th o s e from th e n o v e l L term in u s (p a n e l C t r a c k s 5 -6 ) show

th a t th e n o v e l L term in u s co m p rises in s e r t e d HSV-2 seq u en ce

w ith a te r m in a l ty p e 2 a seq u e n ce , and t h a t th e n o v e l j o i n t

c o m p r ises th e in s e r t e d seq u en ce jo in e d to th e HSV-2 c seq u en ce

v i a a ty p e 2 a se q u e n c e . The two L te r m in i o f RE4 a re

h e t e r o t y p ic , in c lu d in g th e a seq uence (com pare n o v e l term in u s

w ith p a n e l E t r a c k l ) . T h is i s tr u e a l s o o f th e S t e r m in i ,

th e ty p e 1 term in u s b e in g shown i n p a n e l F t r a c k 2 . A

fu r th e r c o m p le x ity in th e genome s tr u c tu r e o f RE4 i s t h a t th e

BamHI ty p e 1 S term in u s , i s o l a t e d from u n iq u e fr a g m e n ts , a l s o

c o n ta in s ty p e 2 S te r m in a l fra g m en ts (p a n e l B t r a c k s 2 - 3 ) .

T h is s u g g e s t s t h a t , l i k e B x l ( 2 8 - 1 ) , a t l e a s t th e ty p e 1 S

term in u s o f RB4 DBA c o n s i s t s o f a m ix tu re o f s t r u c t u r e s

presum ably g en e r a te d by secon d ary r ec o m b in a tio n in v o lv in g th e

r e p e t i t i v e r e g io n s .

R e s t r ic t io n p r o f i l e s o f RE4 and th e r e c le a v a g e ex p er im en ts .

u e s c r ib e d above s u g g e s t th a t th e RE4 genome i s f ix e d i n I and S

in th e Ig arrangem ent (F ig u re C 4 .2 6 ) . For exam ple ,

a u to d e n s ito m e tr ic sca n n in g o f F ig u re C 4.22 t r a c k s R r e v e a le d

th a t HSV-1 H in d l l l m, c o n ta in in g th e ty p e 1 S te r m in u s , i s in

- 145 -

t h r e e - to f o u r - f o ld e x c e s s o v er HSV-1 H in d l l l g , w hich c o n ta in s

th e ty p e 2 S te r m in u s . Two d i s c e r n ib le j o in t fr a g m e n ts , th e

secon d and th ir d bands i n th e Hind l l l p r o f i l e , were un d er

r e p r e s e n te d . The ty p e 1 L term in u s HSV-1 BamHI s was

predom inant o v er th e f a i n t n o v e l term in u s w hich m ig ra ted below

HSV-1 BamHI t , and th e ty p e 1 S term in u s HSV-1 BamHI q was

predom inant o v er th e f a i n t ty p e 2 S term in u s w hich m ig ra ted below

i t . The norm al ty p e 1 j o i n t HSV-1 BamHI k was f a i n t compared

w ith th e n o v e l j o in t o f f a s t e r m o b i l i t y .

The RE4 genome la c k s 10*5 kbp o f DNA in c lu d in g IR^ andJ "I

a t l e a s t 300 bp o f a d ja c e n t seq u e n ce , and h a s an i n s e r t i o n

o f 2 .8 kbp o f HSV-2 DNA a t t h i s l o c a t io n from th e BamHI i - r

( H in d l l l b -n ) r e g io n . M oreover th e r e i s co m p le te t y p e - s p e c i f i c

h e t e r o lo g y betw een IRg and TRg and betw een TR^ and th e j o in t

r e g io n , and th e genome i s f ix e d i n L and S in " th e Ig arran gem en t.

S ev e n te en su b c lo n e s o f RE4 were i s o l a t e d b y 'th r e e c y c l e s o f

p la q u e - p u r i f ic a t io n a t 3 1 ° , and t h e ir genome s t r u c t u r e s a n a ly s e d .

H in d l l l and BamHI p r o f i l e s o f s e v e r a l s u b c lo n e s a re shown in

F ig u re C 4 .2 2 . A l l su b c lo n e s r e t a in th e d e l e t io n and i n s e r t i o n ,

and th e c r o s s o v e r s in and Ug p r e se n t i n RE4. E ig h t o f th e

s u b c lo n e s had r e s t r i c t i o n p r o f i l e s i d e n t i c a l t o th a t o f RE4,

but some more c l e a r l y showed th e f ix e d n a tu re o f L and S

( e . g . su b c lo n c 6 ) , im p ly in g t h a t RE4 i s n o t a pure s t r u c t u r e .

The o th e r n in e su b c lo n e s were p laq u e p u r i f ie d tw id e more

b eca u se s e v e r a l seemed s t i l l to com p rise m ix tu r e s o f genome

s t r u c t u r e s a f t e r th e f i r s t th r e e p laq u e p u r i f i c a t i o n s .

The p r o f i l e s o f m ost o f t h e s e su b c lo n e s are in c lu d e d i n F ig u re

C 4 .2 2 . They a re f r e e in S , w ith th e p o s s ib le e x c e p t io n o f

su b c lo n e 3 , and m ost a re f r e e a ls o in 1 w ith th e e x c e p t io n s o f

17 and p erh ap s 3 , 4 and 5* The d i s t i n c t i o n betw een f ix e d and

f r e e , how ever, i s n o t c l e a r in every c a s e and i t i s l i k e l y t h a t

- 146 -

some s u b c lo n e s may n o t be pure even a f t e r f i v e rou n d s o f p laq u e

p u r i f i c a t i o n . The n in e su b c lo n e s have seco n d a ry c r o s s o v e r s in

r e p e t i t i v e r e g io n s w hich were n o t a n a ly se d f u r t h e r . For exam ple,

s u b c lo n e s 1 , 5 , 7 , 1 5 , 16 and 17 each have a c r o s s o v e r i n HSV-2

BamHI g ’ , r e s u l t i n g in a s m a lle r fra g m en t, w h ich s u g g e s t s th a t

b oth S te r m in i i n t h e s e su b c lo n e s a re ty p e 1 . S u b clon e 1

p o s s ib ly h a s a c r o s s o v e r i n ty p e 1 TR^, s in c e i t . la c k s HSV-1

BamHI s and in s t e a d h a s a te r m in a l fragm en t la r g e r th an HSV-1

BamHI o , a s w e l l a s two o th e r fra g m en ts r e s u l t i n g from th e

la r g e r L te r m in u s . S u b c lo n es 7 , 1 2 , 15 and 16 have a BamHI .

j o in t fragm en t 0 .3 x 10^ la r g e r in m .w t. th an t h a t o f RE4,

p ro b a b ly r e s u l t i n g from a n .e x tr a a seq u en ce in th e j o in t

fragm en t c o n ta in in g in s e r t e d DNA. I t i s co n c lu d ed from t h e s e

i n i t i a l s t u d ie s t h a t in 'R E 4 , a s in B x l ( 2 8 - l ) , th e c o n v e r s io n

o f th e f ix e d p h en otyp e to th e f r e e o c c u r s by a d d i t io n a l

re co m b in a tio n e v e n ts i n r e p e t i t i v e r e g io n s w hich g e n e r a te

t y p e - s p e c i f i c hom ology betw een th e s e r e g ib n s * ., D e t a i le d

r e s t r i c t i o n a n a ly s i s o f th e genomes o f RE4 s u b c lo n e s i s

n e c e s s a r y to more c l e a r l y d e f in e th e secon d ary rec o m b in a tio n

e v e n t s .

- 147 -

DISCUSSION

F ix ed and f r e e reco m b in a n ts

B x l (2 8 -1 ) c o n ta in s a m ixtu re o f a t l e a s t two genome

s t r u c t u r e s d i f f e r i n g i n TR g/lR g. T hese s t r u c t u r e s , and th e

f a c t th a t th e recom b in ant was i n i t i a l l y i s o l a t e d by s i x c y c l e s

o f p la q u e p u r i f i c a t i o n , s u g g e s t th a t th e o r i g i n a l r e c o m b in a tio n

e v e n t produced th e recom b in ant genome s tr u c tu r e shown i n

F ig u re C4. 18. F u rth er in te r m o le c u la r r e c o m b in a tio n p ro b a b ly

th en produced th e s t r u c t u r e s p r e s e n t i n B x l ( 2 8 - 1 ) . I n t r a - or

in te r -m o le c u la r r ec o m b in a tio n e v e n ts c o u ld th en have g iv e n r i s e

to th e s u b c lo n e s . The s t r u c t u r e s p r e s e n t in su b c lo n e 27

presum ably a r o se by s u r v iv a l o f th e i n i t i a l recom b in an t B x l(2 8 )

d u r in g p la q u e p u r i f i c a t i o n , in s im ila r f a s h io n t o th e i s o l a t i o n

o f B x l( 2 8 - 1 ) •

The s u b c lo n e s o f B x l(2 8 -1 ) f a l l in t o two c l a s s e s , th o s e

w hich a re f i x e d in L and th o se in w hich L in v e r t s n o r m a lly .

Each f ix e d su b c lo n e had a fo u r fo ld e x c e s s o f one o r i e n t a t io n o f

L o v er th e o t h e r , and a s im i la r r a t io was o b serv ed f o r L and S

i n RE4. I t i s im p o rta n t to n o te t h a t i n no c a s e was in v e r s io n

o f a segm ent e n t i r e l y a b s e n t , and i t seem s from th e a n a ly s i s

t h a t r e s id u a l in v e r s io n i s a p ro p er ty o f th e f ix e d genome

r a th e r than a r e s u l t o f th e p r e sen ce o f co n ta m in a n ts w ith

seco n d a ry c r o s s o v e r s . The f ix e d p h en otyp e may r e p r e s e n t e i t h e r

p r e f e r e n t i a l r e p l i c a t io n or p r e f e r e n t ia l p a ck a g in g in t o v i r i o n s

o f DNA w ith one o r ie n t a t io n o f L. A n a ly s is o f n u c le a r DNA d id

n o t su p p ort th e l a t t e r (d a ta n o t show n). A l t e r n a t iv e ly , f i x e d

reco m b in a n ts co u ld be th e r e s u l t o f a b ia se d mechanism f o r

m a tu ra tio n o f u n i t le n g t h DNA from c o n c a te m e r s .

The s t r u c t u r e s o f B x l(2 8 -1 ) s u b c lo n e s and o th e r

reco m b in a n ts show t h a t norm al in v e r s io n o f L depends on a

r e g io n o f t y p e - s p e c i f i c hom ology betw een TR^ and IR^. A l l

- 148 -

n o rm a lly in v e r t in g su b c lo n e s p o s s e s s seco n d a ry c r o s s o v e r s i n

TR^ or IR- w hich g iv e r i s e to such hom ology. S u b clo n e 2 9 ,

B x l ( 3 1 - 2 ) 'an d perhaps su b c lo n e 9b show t h a t hom ology betw een th e

a seq u e n c es i s s u f f i c i e n t to a llo w norm al in v e r s io n . I t h as n o t

been d em o n stra ted , how ever, th a t a seq u en ce hom ology i s

n e c e s s a r y f o r norm al in v e r s io n , a s a recom b in an t w ith

hom ologous r e g io n s o f TR^ and IR^ but h e te r o lo g o u s a seq u e n c es

was n o t o b se r v e d . A n a ly s is o f su b c lo n e s o f th e s u b c lo n e s

su p p o r ts th e h y p o th e s is o f hom ology req u irem en ts a f ix e d form

(h e te r o lo g o u s ) gave r i s e to some n o rm a lly in v e r t in g p rogen y

(h o m o lo g o u s), but th e r e v e r s e c o n v e r s io n vras n o t o b se r v e d .

T h is i s e x p l ic a b le in th a t c r o s s o v e r s i n th e f ix e d form can

g e n e r a te hom ology betw een TR and IR^, and t h e s e w ere in d e ed

d e t e c t e d , w hereas a n o r m a lly " in v e r t ih g form ca n n o t g e n e r a te

h e t e r o lo g y s in c e i t d o e s n o t c o n ta in th e n e c e s s a r y s e q u e n c e s .

RE4, i t s u n u su al genome s tr u c tu r e a s id e , i s a n a lo g o u s to

B x l(2 8 -1 ) b ecau se i t i s f ix e d i n L and S i n th e I g o r i e n t a t io n .

I n i t i a l s t u d ie s o f th e s tr u c t u r e s o f RE4 and i t s su b c lo n e s

s u g g e s t t h a t th e hom ology req u irem en t a p p l ie s to b o th L and S ,

s in c e n o rm a lly in v e r t in g progeny p o s s e s s seco n d a ry c r o s s o v e r s

i n T R g/lR g. The s t r u c t u r e s o f reco m b in a n ts w ith two a d ja c e n t

h e t e r o t y p ic a seq u en ces a^ th e j o in t in d ic a t e e i t h e r t h a t

th e q seq u en ce i s c r u c ia l i n segm ent in v e r s io n o r t h a t such an

arrangem ent i s f o r t u i t o u s l y m a in ta in ed . I t i s i n t e r e s t i n g

to n o te th a t th e L term in u s o f D x l(2 ) had a s in g l e ty p e 1

a seq u en ce in about h a l f th e m o le c u le s and an a d d i t io n a l ty p e 2

a seq u en ce in th e o th e r h a l f (o n ly th e form er i s shown in

i i g u r e 0 4 * 1 7 ) .

I t i s proposed from th e se r e s u l t s th a t in v e r s io n o f th e

1 and S segm en ts o f th e HSV genome i s ca u sed by a s i t e - s p e c i f i c

r ec o m b in a tio n ev en t i n , or n e a r , th e a s e q u e n c e . T h is

- 149 -

h y p o th e s is h a s been s tr e n g th e n e d by th e o b s e r v a t io n t h a t

i n s e r t i o n o f a 4 .2 kbp fragm en t c o n ta in in g th e HSV-1 a seq u en ce

in t o th e HSV-1 dPyK gen e ca u sed a d d i t io n a l in v e r s io n s ab ou t

th e n o v e l j o i n t (M ocarsk i e t a l . , 1 980; J . S m ile y , p e r s o n a l

co m m u n ica tio n ). I n s e r t io n o f a fragm en t o f s im i la r s i z e from

e lse w h e r e i n th e genome ca u sed no a d d i t io n a l in v e r s io n s

(M ocarsk i e t a l . , 1 9 8 0 ) .

M odels f o r segm ent in v e r s io n

M odels p r e v io u s ly p rop osed f o r th e m echanism o f in v e r s io n

in v o lv e i n t e r - o r i n t r a - t y p i c r e c o m b in a tio n , and a r e i l l u s t r a t e d

i n F ig u re C 4*27. M odel 1 , w h ich p r o c e e d s v ia p h y s ic a l

s e p a r a t io n o f L and S and t h e i r su b seq u en t r e j o in in g t o g iv e

th e fo u r a rra n g em en ts , h a s n o t been su p p o rted e le c t r o n

m ic r o sc o p ic o r g e n e t ic s t u d ie s (Jacob e t a l . , 1 9 7 9 ) , a lth o u g h

th e r e j o in in g would be s p e c i f i c f o r th e a seq u e n o e . S im i la r ly

m odel 2 , w h ich p r o p o se s t h a t in v e r s io n o c c u r s by in te r m o le c u la r

r e c o m b in a tio n , co u ld in v o lv e a s i t e - s p e c i f i c e v e n t , b u t h a s n o t

th e su p p o rt o f g e n e t ic s t u d ie s (H oness e t a l . , 1 9 8 0 ) . In

m odel in v e r s io n o c c u r s by DNA s y n t h e s i s w ith c o n c u r r e n t

in tr a m o le c u la r r e c o m b in a tio n , but d o e s n o t f i t in w ith th e

c u r r e n t ly a c c e p te d m odel f o r DNA r e p l i c a t i o n . I n tr a m o le c u la ra *

r e co m b in a tio n betw een in v e r t e d r e p e a t s i n l i n e a r o r c i r c u l a r

form (m odel 3B) was o r i g i n a l l y p rop osed a s a n o n - s p e c i f i c

e v e n t , but i n th e l i g h t o f th e above r e s u l t s t h i s i s u n l ik e ly

to be th e c a s e . However, th e m odel can e q u a l ly w e l l

accommodate s i t e - s p e c i f i c re co m b in a tio n a t th e a s e q u e n c e s .

M odel 30 , i s e x p la in e d i n th e le g e n d to F ig u re C 4 .2 7 .

I t h as th e s t r e n g t h s th a t i t accom m odates th e c u r r e n t schem e

f o r DNA r e p l i c a t i o n , and th a t in v e r s io n can be in t e r p r e t e d i n

term s o f a s i t e - s p e c i f i c e v e n t a t th e a s e q u e n c e . One

- 150 -

p r e d ic t io n o f th e m odel i s th a t a l l th e a s e q u e n c e s , and p erh ap s

a d ja c e n t r e g io n s o f th e b and c se q u e n c e s , a re o b l i g a t o r i l y

i d e n t i c a l w ith in a r e p l ic a t e d DNA m o le c u le . T h is f e a tu r e

a r o se b eca u se o f th e r a th e r o v e rr a ted h y p o th e s is t h a t n o t a l l

fo u r genome arrangem ents p a r t ic ip a t e in r e c o m b in a tio n o r

r e p l i c a t i o n , and t h e r e fo r e t h a t g e n e r a t io n o f th e fo u r

arran gem en ts must in v o lv e an o b l ig a to r y e v e n t . T h is h y p o th e s is

i s d is c u s s e d in S e c t io n 2 . B en -P orat e t a l . (1 9 8 0 ) have

r e c e n t ly shown t h a t both arran gem en ts o f th e PRV genome e n te r

t h e n u c le u s , c i r c u l a r i s e , r e p l i c a t e , in v e r t and a re m atured .

Knipe e t a l . (1978) a ttem p ted to make a c a s e f o r o b l ig a t o r y

i d e n t i t y from th e o b s e r v a t io n t h a t i n t e r t y p i c reco m b in a n ts

p o s s e s s hom otypic seq u e n ces in th e a , and p erh a p s a d ja c e n t

r e g io n s o f th e c , seq u en ces bounding Ug . T h is f e a t u r e was

common i n th e reco m b in a n ts d e sc r ib e d i n S e c t io n 2 and i n t h i s

S e c t io n . A l e s s a ssu m p tiv e c o n c lu s io n i s t h a t th e ten d en cy f o r

i d e n t i t y o f t h e s e r e g io n s i s th e r e s u l t o f f a c i l e r e c o m b in a tio n

w ith in r e p e a te d r e g io n s , r a th e r th an th e r e s u l t o f an o b l ig a t o r y

e v e n t i n DNA s y n t h e s i s . A second p ie c e o f e v id e n c e stem s from

th e c la im e d mapping o f an HSV-1 t s n u ta t io n ( t s c 7 5 ) in ea ch a

se q u e n c e . P a r e n t h e t ic a l ly , t h i s d o es n o t d i c t a t e t h a t a

se q u e n c es m ust be o b l i g a t o r i l y i d e n t i c a l , s in c e th e i n i t i a l

m u ta tio n in one a seq u en ce co u ld have been t r a n s fe r r e d to th e

o th e r a seq u e n ces by f a c i l e reco m b in a tio n a t an e a r ly s t a g e .

R e g a r d le s s o f t h i s , P r e s to n ( in p r e s s ) has shown t h a t t h i s

l e s i o n maps w ith in th e Vmw IE 175 gene some 2 kbp away from th e

a seq u en ce i n th e c se q u e n c e . There i s t h e r e fo r e no c o n c lu s iv e

e v id e n c e f o r o b l ig a to r y i d e n t i t y o f th e a s e q u e n c e s . The

o b s e r v a t io n t h a t th e s i z e d i s t r ib u t io n p a t te r n o f th e HSV-1 a

se q u e n c es i s th e same a t th e j o in t and both te r m in i ( S e c t io n 3

o f R e s u lt s ) cou ld a g a in be e x p la in e d by f a c u l t a t i v e i d e n t i t y o f

- 151 -

'these r e g io n s . I*t i s c l e a r from th e work p r e se n te d h ere t h a t

some reco m b in a n ts a re a b le to r e p l i c a t e w ith o u t a req u irem en t

f o r o b l ig a t o r y i d e n t i t y o f a seq u e n c e s ( e . g . su b c lo n e 1 o f

B x l(2 8 -1 ) and RE4). T h ere fo re e i t h e r t h i s i 3 n o t th e

m echanism o f in v e r s io n , or th e s e reco m b in a n ts r e p l i c a t e by an

a l t e r n a t i v e m echanism . The number o f modes o f HSV DNA

s y n t h e s is i s unknown.

The s ta g e a t w hich in v e r s io n o c c u r s d u r in g v ir u s grow th

h as n o t been i d e n t i f i e d , a lth o u g h i s o m e r is a t io n p r o c e e d s to

e q u ilib r iu m a t an e a r ly s ta g e i n p la q u e fo r m a tio n (B en -P o ra t

e t aJL., 1 9 8 0 ) . The e v e n t c o u ld ta k e p la c a e s s e n t i a l l y b e fo r e

th e p r o d u c t io n o f co n c a tem e r s , o r co u ld be p o s t - r e p l i c a t i v e a s

in m odel 3C. I t i s p o s s ib l e t h a t in v e r s io n i s n o t o n ly a

r e s u l t o f r e c o m b in a tio n , but a l s o o f th e mode o f c l e a v in g

u n i t - l e n g t h m o le c u le s from c o n c a te m e r s . I f HSV co n ca tem er s

are produced from a c i r c u l a r in te r m e d ia t e , t h e i r s t r u c t u r e

in th e j o i n t r e g io n s would be d eterm in ed by th e s t r u c t u r e o f th e

j o in t r e g io n s o f th e c i r c u l a r m o le c u le . T h ere fo re th e

mechanism by w hich l i n e a r in p u t DNA c i r c u l a r i s e s would have

a d i r e c t e f f e c t upon con catem er j o i n t s t r u c t u r e .

Mech an ism s f o r genome c i r c u l a r i s a t i o n and m a tu ra tio n

There i s s tr o n g e v id e n c e from e le c t r o n m icro sco p y t h a t

HSV and PRV DNA r e p l i c a t i o n in v o lv e s c o n c a te m e r ic , and p erh a p s

c i r c u l a r , in t e r m e d ia t e s . Jean and B en -P orat (1 9 7 6 ) o b serv ed

l i n e a r PRV DNA m o le c u le s w ith s in g l e s tra n d ed te r m in i and u n i t -

le n g th c i r c l e s in DNA from in f e c t e d c e l l n u c le i i n th e a b se n c e

o f de novo p r o t e in s y n t h e s i s , and p rop osed th a t ex p o su re o f

com plem entary s in g l e stra n d ed te r m in i by an e x o n u c le a se p r e c e d e s

c i r c u l a r i s a t i o n . D e s p ite a s tr o n g b e l i e f in th e te r m in a l

redundancy o f PRV DNA, B en -P orat e t a l . (1 9 7 9 ) w ere u n a b le to

d em o n stra te a te r m in a l r e p e t i t i o n by e x o n u c le a se tr e a tm e n t and

- 152 -

a n n e a lin g i n v i t r o . N e v e r t h e le s s , i t i s p ro b a b le t h a t PRV DNA

i s c i r c u la r a t l e a s t d u r in g th e i n i t i a l s t a g e s o f DNA

r e p l i c a t io n (B en -P o ra t and Veach, 1 9 8 0 ) . On th e o th e r hand,

a lth o u g h HSV-1 DNA i s w ith o u t doubt t e r m in a l ly red u n d a n t, i t

h as n o t been shown th a t c i r c u l a r i s a t i o n i n v iv o o c c u r s by

a n n e a lin g o f exp osed s in g l e stra n d ed t e r m in i . The m a jo r ity o f

B x l (2 8 -1 - 1 ) DNA h as a ty p e 1 a seq u en ce a t th e S term in u s and a

’type 2 a seq u en ce a t th e 1 term in u s, and w ould be u n l ik e ly to

c i r c u l a r i s e by t h i s m echanism s in c e th e HSV-1 and HSV-2 a

seq u e n c e s a r e n o t com plem entary ( S e c t io n .3 o f R e s u l t s ) .

T h e r e fo r e , i f r e p l i c a t i o n p ro ceed s v i a a c i r c u l a r in te r m e d ia t e ,

e i t h e r c i r c u l a r i s a t i o n o c c u r s by an a l t e r n a t i v e m echanism , such

a s b lu n t-e n d l i g a t i o n , o r B x l(2 8 -1 -1 ) DNA c o n t a in s a m inor

p o p u la t io n o f m o le cu le s- w ith hom otypic t e r m in i ( e . g . an e x tr a

ty p e 1 a seq u en ce a t th e ty p e 2 1 term in u s) w hich a lo n e

r e p l i c a t e t o produce f o r unknown r e a s o n s a la r g e p r o p o r t io n o f

n o n - r e p l i c a t in g p rogen y w ith h e te r o ty p ic te r m in i ( i . e . th e

m ajor form o f B x l ( 2 8 - l - l ) genom e). The b i o l o g i c a l p r o p e r t ie s

o f t h i s recom b in an t have n o t been e lu c id a t e d , and o n ly th e

m ajor genome s tr u c t u r e was a n a ly se d , so i t i s n o t a t p r e s e n t

p o s s ib l e to d i s t i n g u i s h betw een th e s e a l t e r n a t i v e s .

The two form s o f c i r c u la r HSV-1 DNA w h ich would be

produced by com plem entary-end and b lu n t-e n d l i g a t i o n , and th e

co n ca tem er s g e n e r a te d th e re fro m , a r e shown i n F ig u r e C 4*28.

I t i s n o te d t h a t b lu n t-e n d l i g a t i o n p ro d u ces co n ca tem er s i n

w h ich a l t e r n a t e j o i n t s may be d i s t in g u is h e d by a c le a v a g e p r o c e s s

to produce u n i t - l e n g t h l i n e a r m o le c u le s . I t i s n e c e s s a r y f o r

b o th segm en ts to in v e r t in d e p e n d e n tly to p rod u ce th e fo u r

a rra n g em en ts . T h is f e a tu r e i s in c lu d e d in m odel 3C i n F ig u r e

C 4 .2 7 , o n th e in c o r r e c t assu m ption th a t th e HSV-1 genome has

two a se q u e n c e s and would th e r e fo r e d i s p la y two d i s s i m i l a r

- 153 -

j o in t s t r u c t u r e s a f t e r corapXementary-end. l i g a t i o n . As shown i n" # T

S e c t io n 3 o f R e s u l t s , th e m a jo r ity o f HSV-1 DNA m o le c u le s

c o n ta in a s i n g l e a seq u en ce a t th e j o in t and t e r m in i , and by

com plem entary-end l i g a t i o n would produce co n ca tem ers w ith

i d e n t i c a l j o i n t s a t a l l l o c a t io n s (F ig u r e C 4 .2 8 ) . C lea v a g e o fr s

u n i t - l e n g t h m o le c u le s from su ch co n ca tem ers would th en

n e c e s s i t a t e a secon d c r i t e r i o n , such a s an ap p rox im ate le n g t h

m easurem ent. The i n i t i a l c h o ic e o f c le a v a g e s i t e i n t h i s c a s e

may be a f a c t o r in in v e r s io n , a s e x p la in e d i n F ig u r e C 4*28.

Jacob e t a l . (1 9 7 9 ) commented t h a t c le a v a g e must be s e p a r a te

from e n c a p s id a t io n i n o rd er to a l lo w p rogen y DNA to e n te r th e

r e p l i c a t i v e p o o l* t h i s would n o t be th e c a s e i f c i r c u la r

monomers a r e r e a d i ly produced from co n ca tem ers by r e c o m b in a tio n .

L adin e t a l . (1 9 8 0 ) have shown t h a t m a tu ra tio n o f PRV DNA i s

d ep en d en t upon o a p s id a sse m b ly . The n a tu r e o f th e c le a v a g e

e v e n t , w h eth er i t i s a d o u b le s tra n d e d c u t o r a s in g l e s tr a n d ed

n ic k a t ea ch end o f th e a seq u en ce fo llo w e d by r e p a ir o f s in g l e

stra n d ed DNA, i s unknown.

F ix ed reco m b in a n ts a re r a r e . In d eed , th e marker r e s c u e

ex p e r im en ts d e s o r ib e d i n S e c t io n 2 w ere aim ed a t g e n e r a t in g

su ch reco m b in a n ts and y e t f a i l e d to do s o . The f ix e d

reco m b in a n ts s tu d ie d r a i s e a number o f q u e s t io n s r e g a r d in g

IISV DNA r e p l i c a t i o n , such a s th e mechanism by w hich su b c lo n e

1 o f B x l ( 2 8 - l ) , t h a t i s B x l( 2 8 - 1 - 1 ) , c i r c u l a r i s e s . More

p r o b le m a t ic a l i s th e way i n w hich th e DNA o f t h i s reco m b in a n t

i s m atu red . W ith r e s p e c t to a se q u e n c e s , c i r c u l a r o r

c o n c a te m e r ic B x l( 2 8 -1 - 1 ) DNA i s s t r u c t u r a l l y i d e n t i c a l to

B x l(3 1 -2 ) DNA, and y e t th e fo rm er h as a f ix e d p h en otyp e and

th e l a t t e r i n v e r t s n o r m a lly . F u rth er a n a ly s e s o f m inor DNA

p o p u la t io n s and o f th e c i r c u l a r s t r u c t u r e s o f th e s e

reco m b in a n ts i s n e c e s s a r y to an sw er su ch q u e s t io n s .

- 154 -

E x p r e s s io n o f B x l(2 8 -1 ) s u b c lo n e s d e le t e d i n TR^/IR^

The r e s u l t s o f ex p er im en ts co n d u cted w ith su b c lo n e s o f

B x l(2 8 -1 ) w hich a r e d e le t e d in e i t h e r TR^ o r IR^ showed th a t

e x p r e s s io n o f o n ly one o f th e two c o p ie s" 'o f Vmw"IE 1 1 0 /1 1 8 i s

s u f f i c i e n t f o r a p r o d u c t iv e i n f e c t i o n in v i t r o . Even"

e x t e n s iv e d e l e t io n o f TR^ r e s u l t e d i n v ia b le v i r u s , a s e v id en ced

by su b c lo n e 22 w hich la c k s a c o n s id e r a b le seq u en ce beyond th e

Vmw IE 118 g e n e . C le a r ly , any o th e r g e n e t ic in fo r m a t io n in

TR j/IR ^ i s n o t r e q u ir e d i n d ip lo id am ounts f o r c o n t in u e d v ir u s

r e p l i c a t io n in t i s s u e c u l t u r e . RE4 h a s a d e l e t io n e x te n d in g

from th e j o i n t , th ro u g h IR-^, and a t l e a s t 300 bp in t o U^,

but th e p o ly p e p t id e s in d u ced by t h i s recom b in an t have n b t y e t

been exam ined . The o b s e r v a t io n t h a t a l l r eco m b in a n ts exam ined,

in c lu d in g th o s e w ith d e le t e d s e q u e n c e s , have r e t a in e d a se q u e n c e s

a t th e j o i n t and te r m in i may be a r e s u l t o f a req u irem en t f o r ,

or r e g e n e r a t io n o f , th e a se q u e n c es i n DNA r e p l i c a t i o n .

D e le t io n s i n TRg/lRg a n a lo g o u s to th o s e i n TR j/lR ^ have n o t been

o b se r v e d , p erh ap s b ecau se o f a req u irem en t foi* or" g r e a t e r

s e l e c t i v e ad v a n ta g e o f f u n c t io n a l d ip lo id y o f TRg/lRg i n

p r o d u c t iv e i n f e c t i o n , or b eca u se o f th e m echanism o f d e l e t io n

w hich i s a t p r e s e n t unknown.

There a re s e v e r a l p o s s ib l e e x p la n a t io n s o f th e d ip lo id y

f o r T R j/IR ^ and TRg/IRg d is p la y e d by a l l f i e l d i s o l a t e s

exam ined to d a t e . Two o u t o f tw e n ty B x l( 2 8 -1 ) s u b c lo n e s were

d e le t e d i n TR or IR^, and y e t no e q u iv a le n t d e l e t io n s have been

d e te c te d in o v er a hundred i n t e r t y p i c reco m b in a n ts i s o l a t e d so

f a r i n G lasgow . As w ith n a tu r a l i s o l a t e s , th e fre q u e n cy w ith

w hich d e l e t io n s o ccu r or a re i s o l a t e d in th e l a t t e r c a s e m ust

be lo w e r . The d e le t e d genom es r e s u l t e d from reco m b in a n ts w hich

a re c o m p le te ly h e te r o lo g o u s f o r one o r b oth s e t s o f r e p e a t s .

No o th e r recom b in an t or f i e l d i s o l a t e has been shown to p o s s e s s

- 155 -c o m p le te ly h e t e r o ty p ic d ip lo id r e g io n s ; th u s , i f d e l e t io n s a re

n a t u r a l ly b e in g g e n e r a te d , t h e i r fr e q u e n c y m ust be so low a s to

have escap ed i s o l a t i o n in th e r e l a t i v e l y sm a ll number o f

f i e l d i s o l a t e s exam ined to d a t e . I t i s n o t known why

h e te r o ty p ic reco m b in a n ts w ith d e l e t io n s in TR^/IR-^ were so

r e a d i ly i s o l a t e d , and n e i t h e r i s i t known w h e th e r " th is i s

p r im a r ily a r e s u l t o f th e fr e q u e n t g e n e r a t io n o f d e l e t io n s o r

o f t h e i r su b seq u en t s e l e c t i v e a d v a n ta g e . I t sh o u ld be n o ted

th a t d e le t e d TR or IR^ seq u e n c e s can be r e g e n e r a te d in

hom otypic genombs by r e c o m b in a tio n , w h ereas t h i s i s n o t

p o s s ib le in h e t e r o ty p ic r ec o m b in a n ts .

I f v ir u s e s w ith d e l e t io n s i n th e genome were a t a

s e l e c t i v e d isa d v a n ta g e f o r grow th , n a t u r a l ly o c c u r r in g d e l e t io n

m utants would n o t s u r v iv e . The d e t a i l e d grow th c h a r a c t e r i s t i c s

o f d e le t io n m u tan ts have y e t to be i n v e s t ig a t e d i n v i t r o and

in v i v o . I t i s p o s s ib l e t h a t in c r e a s e d g en e d o sa g e r e s u l t i n g

from d ip lo id seq u e n c es c o n f e r s an a d v a n ta g e i n e s t a b l i s h i n g an

i n f e c t i o n a t low m u l t i p l i c i t i e s , o r i n c e l l t y p e s n a t u r a l ly

r e s i s t a n t to v ir u s i n f e c t i o n o r r e p l i c a t i o n , and t h i s m ight

be p a r t ic u la r ly im p o rta n t i n th e n a tu r a l h o s t .

I t i s a l s o p o s s ib l e t h a t th e p r e s e n c e o f in v e r t e d d ip lo id

r e g io n s , o r th e e x p r e s s io n o f g e n e s c o n ta in e d in them , i s

e s s e n t i a l f o r an u n id e n t i f i e d f u n c t io n o f th e Y ir u s , f o r

exam ple i n th e p r o c e s s e s o f la t e n c y o r c e l l u l a r tr a n s fo r m a t io n .

An u n l ik e ly a l t e r n a t i v e i s t h a t th e p r e s e n c e o f d ip lo id r e g io n s

i s o f no c r u c ia l con seq u en ce to HSV, but m erely r e f l e c t s a

p r o p e n s ity o f DNA to r e p l i c a t e w h erever p o s s i b l e .i

Genome s tr u c tu r e o f RE4

F in a l ly , i t i s w o rth w h ile com m enting on th e e x tr a o r d in a r y

genome s tr u c tu r e o f RB4. T h is v ia b le recom b in an t la c k s 10*5 kbp

o f DNA, in c lu d in g IKL and a s h o r t a d ja c e n t r e g io n o f UL.

- 156 -

2 .8 kbp o f HSV-2 DNA have been in s e r t e d i n t o th e p o s i t i o n o f

th e d e l e t i o n , an e v e n t w h ich presum ably o ccu rred i n th e same

c e l l a s th e i n i t i a l re co m b in a tio n e v e n t , r e n d e r in g th e genome

h e t e r o t y p i c a l l y " d ip lo id 1* f o r t h i s r e g io n o f U^. I t i s n o t

y e t known w h eth er th e in s e r t e d seq u e n c e s a r e f u n c t io n a l i n RE4,

even to th e p o in t o f f u l f i l l i n g a f u n c t io n o f d e le t e d

se q u e n c e s . I t i s o f i n t e r e s t to n o te t h a t th e in s e r t e d -

seq u en ce a lm o s t c e r t a i n l y c o n t a in s u pstream seq u e n c e s and

p erh a p s a p a r t o f th e t r a n s c r ib e d seq u en ce f o r th e gen e c o d in g

' fo r HSV—2 MOP, a m ajor l a t e p o ly p e p t id e . T h is i n t e r e s t i n g

d e le t e d reco m b in a n t w hich i s a l s o f ix e d i n both segm en ts

f u r n is h e s o p p o r tu n ity f o r f u r t h e r s tu d y .

Figure C4.1

Structures of the joints and long termini of Bxl(28-1).

A 1 tsB BamH I k“ 2 B x l(2 E P l) BamH I m ajor j o in t

3 B x l (2 8 -1 ) BamH I m inor j o in t4 t s l BamH I g

D ig e s te d w ith S s t I ; 2 $ a g a r o se g e l .

B 1 tsB BamH I k2 B x l (2 8 -1 ) BamH I ma^or j o in t3 t s l - BamH I g and g

D ig e s te d w ith Sma I ; 7 . 5 % p o ly a cr y la m id e g e l«

D. 1 tsB BamH I ^ t2 t s l BamH I vw3 B x l(2 8 -1 ) BamH I major typ e 2 lo n g term in u s4 B x l(2 8 -1 ) BamH I m inor typ e 1 lo n g te r m in u s ,

w ith HSV-2 Bamll I &

D ig e s te d w ith S s t I ; 2^ a g a r o se g e l .

D 1 tsB BamH I s t2 t s l BamH I vw3 B x l(2 8 -1 ) BamH I m ajor ty p e 2 lo n g term in u s

D ig e s te d w ith Sma I ; 7 .5 # p o ly a c r y la m id e g e l .

S s t I or Sma I r e s t r i c t i o n fra g m en ts o f j o in t o r lo n g te r m in a l fra g m en ts a re in d ic a t e d to th e l e f t (HSV-1) and r ig h t (HSV-2) o f each p a n e l .X d e n o te s a genome te r m in a l fra g m en t, and J th e Sma I fra g m en t sp an n in g th e ju n c t io n betw een th e two tandem a se q u e n c e s in HSV-2 BamH I g .

With r e s p e c t to th e t e x t , th e term s lo n g ter m in u s and 1 te r m in u s , and s h o r t term in u s and S te r m in u s , a re e q u iv a le n t .

CM OO^f

CM 00 lOi i i

co

O)I I 1

< COO Qu-I ll XCO ” “ 5

lO CO

h- oo a> ° £■ i i i i

O Q-

h - CDi i

F igure C4.2

Structures of the short termini of Bxl(28-l).

A 1 t s B he pit I h2 J3xl(28-rl) hcoK I r ig h t hand S term in u s3 t s l . hooK I m

D ig e s te d w ith S s t I ; 1 .5 •'* a g a r o se g e l .

B As panel A but digested with Kpn I .

C 1 tsB Hind I I I m" 2 Bxl ( 2 8 -1 ) Bgl~~II l e f t hand S term in u s

3 t s l Bffl II~ra

D ig e s te d w ith S s t I ; 1 .5 /* a g a r o se g e l .

D As p a n e l C b u t d ig e s t e d w ith Kpn I .

h 1 tsB BamH I £gi~ 2 t s l BamH I r .

3 TqI BamH I s t4 B x l (2 8 -1 ) BamH I type 1 short terminus,

w ith HSV-2 BamH I r^5 B x l (2 8 -1 ) BamH I type 2 sEort terminus6 tsl-Bam H I u

D ig e s te d with Sma I ; 7 .5 /* polyacrylamide gel.

S s t I , Knn I o r Sma I r e s t r i c t i o n fra g m en ts o f s h o r t te r m in a l fra g m en ts a re in d ic a t e d to th e l e f t (HSV 1 ' ana r ig h t(H S V -2 ) o f each p a n e l . In p a n e ls A and o n ly th o se S3 t I fra g m en ts mapping i n th e BamH I te r m in a l fra g m en t a re in d ic a t e d .A u e n o te s a genome te r m in a l fra g m en t.

I o

l

To

QX

^

Figure C4.2

Structures of the short termini of Bxl(28-1).

A 1 t s B Bcoit I k2 B x l (28^*1) BcoK I r i g h t hand S term in u s3 t s l . EcoH I m

D ig e s te d w ith S s t I ; 1 .5 '* a g a r o se g e l .

B As p a n e l A h ut d ig e s t e d w ith Kpn I .

C 1 tsB Hind I I I m~ 2 B x l (2 8 -1 ) B g l~ II l e f t hand S term in u s

3 t s l B g l I l ” m

D ig e s te d w ith S s t I ; lo5 /* a g a r o se g e l .

JJ As p a n e l C but d ig e s t e d w ith Kpn I .

K 1 tsB BamH I pp.~ 2 t s l BamH I r .

3 t s l BamH I s t4 B x l(2 8 -* l) BamH I ty p e 1 s h o r t term in u s ,

w ith HSV-2 BamH I r s5 B x l(2 8 -1 ) BamH I ty p e 2 sE o r t term in u s6 t s l - BamH I u

D ig e s te d w ith Sma I ; 7*5/* p o ly a c r y la m id e g e l .

S s t I , Kpn I or Sma I r e s t r i c t i o n fra g m en ts o f s h o r t te r m in a l fra g m en ts a re in d ic a t e d to th e l e f t (HSV-1) and r ig h t (liSV -2) o f each p a n e l . In p a n e ls A and 0 o n ly th o se S s t I fra g m en ts m apping i n th e BamH I te r m in a l fragm en t a re i n d ic a t e d . j£ u e n o te s a genome te r m in a l fr a g m en t.

CO

CM

I

XLO

■

QX

h

P igure C4» 3

P r e v io u s ly p u b lish e d recom b in ant genome s t r u c t u r e s .

HSV-1 seq u e n c es a re in d ic a t e d above and HSV-2 seq u e n ces below

th e r e p r e s e n t a t iv e genome i n each c a s e . C r o ss -h a tc h in g

d e n o te s th e r e g io n o f u n c e r ta in ty i n c r o s s o v e r p o s i t i o n .

R e fe r e n c e s i

B x l ( 2 o - l ) , 8 x 1 (3 1 -2 ) and 8 x 1 (2 ) - P r e s to n e t a l . (1 9 7 8 ) ,

RS6 - Stow and W ilk ie (1 9 7 8 ) ,

8 x 6 (1 7 -6 ) - iviarsden e t a l . (1 9 7 8 ) ,

10.35-2 - C hartrand e t a l . (1 9 8 1 ) ,

Sach recom b in an t in v e r t s n o rm a lly in L and S e x c e p t B x l ( 2 8 - l ) ,

i n w hich L i s f ix e d in th e o r ie n t a t io n p r e s e n t in th e P and

Ig genome a rra n g em en ts .

.COd

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r fO

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X00

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cox

00

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CMi

00

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11,-Ture 04>4

R e s t r i c t io n p r o f i l e s o f in v iv o ^2P - la b e le d DNA

from s u b c lo n e s o f B x l (2 8 -1 ) f o r BcoR I (A ,B ) and

Hpa I (C ,D ). The su b c lo n e numbers a r e g iv e n a t

th e top o f th e t r a c k s , and a l s o in c lu d e d a re

HSV-1 tsB ( T l ) , HSV-2 t s l (T2) and B x l (2 8 -1 ) (R ) .

R e s t r i c t io n fra g m en ts are in d ic a t e d to th e l e f t

(iiS V -l) and r ig h t (R3V-2) o f each p a n e l .

0 .4 # a g a r o se g e l s were u se d .

A n R 1 2 102729 T2 B T1 R 5 9 11 14 22 T2

* ®d _ — . . _ _ r

de f g B » S - - ? 5 * g . h i :.

• * mm

I *m

m *•

l

C T1 R 1 2 10 2729 T2 D T1 R 5 9 11 14 22 T2abed

Pigure C4.5

R e s t r ic t io n p r o f i l e s o f in v iv o ^2P - la b e le d DNA

from s u b c lo n e s o f B x l (2 8 -1 ) f o r Kpn I (A ,B) and

Bamll I (Cf D ). The su b c lo n e numbers a re g iv e n a t

th e to p o f th e t r a c k s , and a l s o in c lu d e d a re

liSV-1 tsB ( 1 1 ) , HSV-2 t s l (1 2 ) and B x l ( 2 8 - l ) (R ).

R e s t r i c t io n fra g m en ts a r e in d ic a te d to th e l e f t

(HSV-1) and r ig h t (HSV-2) o f each p a n e l .

0#7r* and 1 .0 ^ a g a r o se g e l s were u se d .

A

abedeJgj j

T1R 1 2 10 27 29 T2 B T1 R 5 9 11 14 22 T2

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m — n o P P *■

rci ms+l u —

vwxyz

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C n R 1 2 102729T 2 D

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; gWi ^ ft* hijfc. k

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r - = = - - :^ W « i « M

I

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Fig u r e C4.6

G ross s t r u c t u r e s o f B x l (2 6 -1 ) s u b c lo n e s . B lack

r e p r e s e n t s HSV-2 DNA, and w n ite HSV-1 DMA, u s in g

th e c o n v e n t io n a l r e p r e s e n ta t io n o f th e 1ISV genom e.

H egion s o f u n c e r ta in ty o f c r o s s o v e r have been

o m itte d s in c e p r e c i s e d e t a i l s a r e g iv e n in

F ig u re C 4 .1 7 . A c r o s s i n d i c a t e s d e le t e d s e q u e n c e s .

The in v e r s io n p ro p e r ty o f L i s n o te d , and the

su b c lo n e number i s g iv e n on th e r i g h t .

FIXED

FREE

9

m

1. 2 . 6 . 12.16.25

lO — E U

3.4.7.8. 21.26.29

11

10

1— E 9

a E 2 2

27 as Bx 1(28-1)

P ig u re jC iil

Structures of the joints of Bxl(28-1) suhclones

A 1 tsB BamH I k2 J s l BaraH I g3 su b c lo n e 1 BamH I j o in t4 su b c lo n e 5 BamH I j o in t5 su b c lo n e 9 BamH I j o in t6 su b c lo n e 29 BamH I j o in t

D ig e s te d w ith S s t I ; 2 $ a g a r o se g e l .4

B 1 tsB BamH I k"" 2 su b c lo n e 10 Bamli I j o in t

3 t s l BamH I4 t s l BamH I g

D ig e s te d w ith S s t I ; 2 $ a g a r o se g e l .

G 1 tsB BamH I k~ 2 t s l BaraH I g

3 su b c lo n e 1 BaraH I j o in t4 su b c lo n e 9 BamH I j o in t3 su b c lo n e 29~BamiI I j o in t

In v i v o - la b e le d DMA c le a v e d w ith Sraa I ; 7 ,3 /* p o ly a c r y la m id e g e l .

S s t I or Sma I r e s t r i c t i o n fra g m en ts o f j o i n t fra g m en ts a re in d ic a te d to th e l e f t (HSV-1) and r i g h t (lISV-2) o f each p a n e l .

i - cvjco^f in co h* ooi i i i i

I I l ii i i i i i i i i

O l < 0Q(J LU LL X U —

h- CO

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COCM

<1

figure C4»8

Structures of the joints of Bxl(28-1) subclones.

A 1 t sB BamH I k~ 2 su b c lo n e 1 BaraH I j o in t

3 su b c lo n e 9 BamH I j o in t4 t s l BamH I g

D ig e s te d w ith Sma I ; 7 . 5 ‘* (u p p er) and 1 5 & ( lo w e r ) p o ly a c r y la m id e g e l s . * -

B 1 tsB BamH I k2 su b c lo n e 29~BaraH I la r g e r j o in t3 su b c lo n e 29 SaraH I s m a lle r j o in t4 t s l BamH I g

D ig e s te d w ith Sma I ; (u p p er) and 1 5 # (lo w e r )p o ly a c r y la m id e ~ g e ls .

0 1 tsB BamH I k2 su b c lo n e 14 BamH I / Hpa I j o in t3 t s B Hna I m

D ig e s te d w ith Sma I ; 7 . 5 ‘* p o ly a c r y la m id e g e l .

D 1 tsB BamH I k2 su b c lo n e 10 BaraH I j o in t3 t s l Damn I g

D ig e s te d w ith Sma I ; 7 .5 # p o ly a c r y la m id e g e l .

Sma I r e s t r i c t i o n fra g m en ts o f j o in t fra g m en ts a re in d ic a t e d to th e l e f t ( HSV-1) and r ig h t (I1SV-2) o f eacn p a n e l .

A 1 2 3 4 B 1 2 3 4 C 1 2 3 D l 2 3

M «

figure 04.9

Structures of the long termini of Bxl(28-1) subclon

A 1 tsB Bamh I s2 su b c lo n e 1 Bamh I3 su b c lo n e 1 Bamh I

wi th iiSV-24 su b c lo n e 5 Bamli I5 su b c lo n e 5 Bamh I

w ith “ iiSV-26 su b c lo n e 9 Bamli I

w ith “ h3V-27 su b c lo n e 29 Bamh

w ith HSV-28 su b c lo n e 29 ilSV-29 su b c lo n e 29 Bamil

w ith lioV-210 B xl (3 1 -2 ) Bamil I

w ith 113 V-211 t s l Bamli I uvw12 T s l Ba;uh I wx

typ e 2 lo n g term in u s ty p e 1 'lo n g term in u s , Bamil I xtyp e 2 lo n g term in u s typ e 1 lo n g term in u s, Bamh I xtyp e 1 lo n g term in u s , BamH I x

I typ e 2 ~ lo n g term in u s , BaraH I u Bamil I x

I typ e l~ lo n g term in u s , Bamil I x

typ e 1 lo n g term in u s,Bamh I x

D ig e s te d w ith S s t I ; a g a ro se g e l .

tsB BaraH t s l Bamil

s ivw

su b clon e 9 5amn I type 2 lo n g term inus su bclone 9 Bamh I type 1 lo n g term inus su b clon e 29 Bamh I type 2 lo n g term inus 3Ubclone 29 Bamh I type 1 lon g term inus

D ig e s te d w ith S s t I ; 2 ‘« a g a ro se g e l . Term ini in t r a c x s 3 -6 were i s o l a t e d v ia th e u n iq u e lipa I lo n g te r m in a l fr a g m e n ts .

C 1 t s l Bamli I uvw2 su b c lo n e 10 Bamh I typ e 2 lo n g term in u s3 su b e lo n e 14 Bamh I ty p e 2 lo n g term in u s

D ig e s te d w ith S s t I ; a g a r o se g e l .

3 j t I r e s t r i c t i o n fra g m en ts o f lo n g te r m in a l fragm en ts a r e in d ic a t e d to the l e f t (h o V -l) and r ig h t (HSV-2) o f each p a n e l .A u e n o te s a genome term in a l fra g m en t.

to 1m 1 1

1

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I

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XI COI I

CO x

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CD X<1

Pigure C4.1Q

Structures of the long termini of Bxl(28-1) subclones,

A 1 tsB BamH I s2 subcTone 9 BamH I ty p e 1 lo n g term in u s ,

w ith IiSV-2 BamH I x3 su b c lo n e 29 BamH I ty p e 2~"long term in u s4 su b c lo n e 29 Bamll I ty p e 1 lo n g term in u s5 t s l BamH I vw

'6 su b c lo n e 1 BamH I ty p e 2 lo n g term in u s

n ig e s t e a w ith Sma I ; 7•5!* (u p p er) and 15> (lo w e r )p o ly a c r y la m id e g e l s . * -

B 1 tsB Bamll I k.2 t s l BamH I vw3 su b c lo n e 10 BamH I typ e 2 lo n g term in u s4 su b c lo n e 14 Bamll I ty p e 2 lo n g term in u s

D ig e s te d w ith Sma I ; 7*5# p o ly a c r y la m id e g e l .

0 1 tsB Hoa I m2 su b e lo n e 22 typ e 2 Hpa I d e le t e d lo n g term in u s

D ig e s te d w ith Bamll I / Sma I ; 7.51* p o ly a c r y la m id e g e l .

D 1 tsB BamH I s~ 2 TBB Hpa I nf~

3 su b c lo n e 22 typ e 1 Hpa I lo n g term in u s4 su b c lo n e 22 ty p e 2 Hpa I d e le t e d lo n g term in u s3 t s l Hpa I g6 Tsl Hpa I x7 t s l BamH I vyy

D ig e s te d w ith Sma I ; 2 ‘t a g a r o se (u p p er) and 7 .5 ^ p o ly a c r y la m id e ( l o w e r ) - g e l s .

Sma I r e s t r i c t i o n fr a g m en ts o f lo n g te r m in a l fra g m en ts a re in d ic a t e d to th e l e f t (hSV -1) and r ig h t (HSV-2) o f each pari e l .A d e n o te s a genome te r m in a l fra g m en t.

2 3

4 5

6 7 1

1 1 * 11 II $ 1

•m ! i

> • ? !m iQ

CM f- 1

o o c

cm corf^ I VCO ^CM f l

«- |00 < COO Oku 1

f N i » •I 1 I I I !

CJ Q I O - “3

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1C•V

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tO XXIs-I 0) o * -

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U O I O — >I-J

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figure C4.ll

Structures of the short termini of Bxl(28-1) subclones.

1 tsB BamH I2 t s l BamH I u3 su b c lo n e 1 Hamil I s h o r t te rm in u s ,

w ith HSV-2 BamH I r4 su b c lo n e 5 BamH I s h o r t te r m in u s ,

with""il3V-2 BamH I s5 su b c lo n e 9 BamH I s h o r t te r m in u s ,

w itlT iISV -2 BaraH I r6 su b c lo n e 29 BamH iI s n o r t term in u s7 B x l (3 1 -2 ) BamFTl :sh o r t term in u s ,

w ith HSV-2 Bamll I rco t s l BamH I pq t s l BamH 1 r

D ig e s te d w ith S s t I ; 2 # a g a r o se g e l*

B 1 t s l Bamll I r2 t s B BamH I3 su b c lo n e 10 Bamll I s n o r t term in u s4 su b c lo n e 14 BamH I s h o r t te r m in u s ,

w ith HSV-2 BamH I r5 tQ l BamH I u ~ ~

D ig e s te d w ith S s t I ; 2 fa a g a r o se g e l .

C R e s t r i c t io n p r o f i l e s o f in v iv o ^2P - la b e le d~ DNA from s u b c lo n e s o f BXIT28-T7 fo r BamH I ,

in th e r e g io n o f th e v e r y sm a ll fr a g m e n ts .The su b c lo n e numbers are g iv e n a t th e top o fth e t r a c e s , and a l s o in c lu d e d a re HSV-1 tsB (11 )and H3V-2 t s l ( 1 2 ) . R e s t r ic t io n fra g m en ts are in d ic a te d to th e l e f t (HSV-1) and r ig h t (H SV -2). A p o ly a c r y la m id e g e l was used •

S s t I r e s t r i c t i o n fra g m en ts o f s h o r t te r m in a l fragm en ts a re in d ic a t e d to th e l e f t (HSV-1) and r i g h t (HSV-2) o f p a n e ls A and B.X d e n o te s a genome te r m in a l fra g m en t. A lso marked in p a r e n tn e se s in p a n e l A a re S s t I 2 an(i 4. °?HSV-2 BamH I v , tn e lo n g ter m in u s .

figure C4.12

LEPT HAND TWO PANELS

S tr u c tu r e s o f th e s h o r t te r m in i o f B x l(2 8 -1 ) s u b c lo n e s .

A 1 tsB BamH I pg_2 t s l BamH I r3 t s l BamH I s t4 t s l BamH I u3 su b e lo n e 1 BamH I sn ^ rt te rm in u s ,

w ith HSV-2 BamH I r s6 su b c lo n e 5 BamH I s h o r t te r m in u s ,

with~’HsV-2 BamH I s7 su b c lo n e 9 BamH I s h o r t te r m in u s ,

w ith HSV-2 Bamu I r8 su b c lo u e 29 Bamii I~sITort term in u s

D ig e s te d w ith Sma I ; 7 .5 /* p o ly a c r y la m ia e g e l .

B 1 t s l BamH I r2 tsB Bamli I £9.3 su b c lo n e 10 Bamli I s a o r t term in u s4 su b c lo n e 14 Bamli I s n o r t term in u s 3 t s l Bamll I u

D ig e s te d w ith Sma I ; 7 . 9 p o ly a c r y la m id e g e l .

Sma I r e s t r i c t i o n fra g m en ts o f s h o r t te r m in a l fragm en ts a r e in d ic a t e d to th e l e f t ( ilS V -l) ana r i g h t (HSV-2) o f th e p a n e l3 .X d e n o te s a genome te r m in a l fra g m en t.

RIGHT HAND TWO PANELS

A u torad iograp h o f n i t r o c e l l u l o s e b l o t s t r i p s c o n ta in in g Kpnl or BamHI r e s t r i c t i o n fra g m en ts o f su b c lo n e 14 (S lJT or 2<TX322) LNA., to w h ich n ic k t r a n s la t e d BNA p ro b es had been h y b r id is e d .1 1The p ro b es w ere: HSV-1 DNA (H ), and a m ixtu re o f p la sm id s c o n ta in in g liS V -l BamHI k and. HSV-2 BamHI g ( J ) . HSV-1 DNA h y b r id is e d s t r o n g ly ..to HSV-1 fra g m en ts, in th e recom b in an t genom es, and to a l e s s e r e x t e n t to HSV-2 fr a g m e n ts . An e x p la n a t io n o f th e bands to w hich th e J probe h y b r id is e d i s g iv e n (NLT - n o v e l L term in u s; HI -.H SV -1 fragm en t;H2 - HSV-2 fra g m en t; S . - S segm en t, s in c e th e su b c lo n e genom es have no Kpnl s i t e s in S ) .

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in j u r e C4.13

Structures of the joints and termini of Bxl(31-2).

A i tsB BamH I s t7 2 B x l (3X72) BamH I j o i n t ,

w ith o th e r fragm ents..3 B x l (3 1 -2 ) BamH I s h o r t term in u s and ty p e 2

la r g e r lo n g te rm in u s , w ith HSV-2 BamH I4 B x l (3 1 -2 ) HSV-2 BamH I s -----5 B x l£ 3 1 -2 ) BamH I ty p e l~ lo n g ter m in u s ,

w ith HSV-2 BamH I x6 t s l BamH I vw ~ ~

D ig e s te d w ith S s t I ; 2>« a g a ro se g e l .

B As f o r psu*el A but d ig e s t e d w ith Sma I ; 7 .5 # p o ly a c r y la m id e g e l .

0 1 t s B Bamii I s t2 t s l BamH I vw3 B x l (3 1 -2 ) BamH I type 2 lo n g term in u s4 B x l (3 1 -2 ) Bamli I typ e 1 lo n g term in u s5 D x l(2 ) BamH I lo n g term in u s from th e r ig h t

Haiid end o f L6 D x l(2 ) BamH I lo n g term in u s from th e l e f t

hand end o f L7 D xl (2 ) Bamii I s h o r t- te r m in u s from th e

r ig h t hand end o f S8 D x l(2 ) BamH I s h o r t term in u s from th e

,X eft hand end o f S9 tsB BamH I pq

10 t s l BamH I u

Fragm ents fo r tracK s 3 -3 i s o l a t e n from la r g e r u n iq u e fr a g m e n ts . D ig e s te d w ith S s t I ; 255 a g a r o se g e l .

D 1 KSV-1 S s t I lo n g term in u s~ 2 t^ 6 S s t 1 lo n g term in u s

3 B x l (3 1 -2 ) S s t I ty p e 2 la r g e r lo n g term in u s4 B x l i3 1 - 2 ) S s t I ty p e 1 lo n g term in u s5 D x l(2 ) S st~ T ~ Ion g term in u s from th e r ig h t

hand end o f L6 D x l(2 ) S s t I lo n g term in u s from th e l e f t

Hand end o f L7 t s 6 S s t I lo n g term in u s

Ss t I fra g m en ts i s o l a t e d from la r g e r fr a g m e n ts , w hich were u n iq u e f o r t r a c e s 3 - 6 . D ig e s te d w ith Sma I ;7.3,-® p o ly a c r y la m id e 0 e l

ites t r i e t io n fra g m en ts o f Bamii I te r m in a l fra g m en ts a re in d ic a t e d to th e l e f t (HSV-1) and r ig h t (HSV-2) o f th e p a n e ls e x c e p t in p a n e l 0 where lo n g te r m in a l fra g m en ts a re in d ic a t e d on th e l e f t and s n o r t te r m in a l fra g m en ts

w ith nSVr2 fra g m en ts in p a r e n th e s e s , x d e n o te s a genome UH’M inal fT agm cnt.

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Figure C4.14

Structures of the joints and termini of Bx6(17-6).

1 tsB2 tsB3 tsB4 5 x 65 Bx66 Bx6

7 t s l8 T s l9 t s l

10 t s l

A 1 ta l i2 B x l( 3 1 -2 )3 Bx 6 (1 7 -6 )4 t s l -

In v iv o ^ P ~ la b e le d j>i>[a d ig e s t e d w ith BamH I ; If* a g a r o se g e l .

B 1 tsB BamH I s t |sB BamH I s tsB BamH I k

BamH I j o i n t B x 6 ( l7 -6 ) Bamii I s h o r t term in u s B x 6 ( l7 -6 ) Bamii I lo n g te rm in u s ,

w ith H3V-2 BamH I w

t s l Bamli I vw t s l Bamii I x

D ig e s te d w ith S s t I ; 2f» a g a r o se g e l

G 1 tsB Bamii I k2 5 x 6 ( 1 7 -6 ) Bamii I jo in t3 t 3 l - BamH I~ g

D ig ested w ith Sma I ; 7 * 5 # p olyacry lam id e g e l .

D 1 tsJi Bamii I s2 J sB Bamii I ^3 Bx6 ( 1 7 -6 ) BamH I s h o r t term in u s4 B x 6 (1 7 -6 ) BamH I lo n g te rm in u s ,

w itE H3V-2 BamH I w5 t s l BamH I u ~6 t s l Bamii i vw7 t s T BamH I x

D ig e s te d w ith Sma I ; 7 « 5 ‘* p o ly a c r y la m id e g e l .

HSV-1 and HSV-2 r e s t r i c t i o n fragm en ts a re in d ic a t e d to th e l e f t and r i g h t , r e s p e c t i v e l y , o f each p a n e l .In p a n e l A th e y r e f e r to th e Bamll I d i g e s t o f HSV-1 and HSV—2~DJMA, and in p a n e ls B-D to th e S 3 t I o r Sma I d i g e s t o f Bauni I j o i n t fr a g m e n ts .

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Figure C4.15

Structures of the joints and termini of HS6 and HI3-2.

A 1 tsB BamH I k2 TsB BamH I a3 HS6 BamH I j o in t4 KS6 BamH I lo n g term in u s5 t s l BamH I g6 T s l BamH I u7 t s l BamH I vw

D ig e s te u w ith S s t I ; 2>'« a g a ro se g e l .

B As f o r p a n e l A but d ig e s t e d w ith Sma I ; 7 .5 # p o ly a c r y la m id e g e l .

C 1 tsB BamH I k~ 2 t s l BamH I g

3 RT3-5"T?amIl I la r g e r j o in t4 H13-2 BamH I s m a lle r j o in t5 HI3—2 BamH I typ e 2 lo n g term in u s6 H 13-2 Bamli I ty p e 1 lo n g term in u s ,

w ith HSV-2 BamH I x7 t s B BamH I s ”* *"8 t s l BamH I vw

D ig e s te d w ith Sma I ; 7 .5 # (u p p er) and 1 5 # ( lo w e r )p o ly a c r y la m id e g e l s . - -

S s t I or Sma- I r e s t r i c t i o n fra g m en ts o f BamH I " jo in t fra g m en ts a r e in d ic a t e d to th e le ft^ T E S V -l) and r ig h t (liSV -2) o f each p an el.

t - CM CO If)

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Figure C4.16

Structures of the joints and termini of Dx 1(2)-.

A 1 tsB BamH I k2 t s l) Bamk I k3 B xl (2T""BamH~I j o in t4 s u b c lo n e o f su b c lo n e 2 o f B x l( 2 8 -1 ) BamH I j o in t5 t s l Bamll I g

D ig e s te d w ith Sma I ; 7 .5 # (u pp er) and 1 5 # ( lo w e r ) p o ly a c r y la m id e g e l s . - _ « „

B 1 t s B S s t I C and D ( i s o l a t e d )2 Bxl(2j~BamH I s h o r t term in u s from th e

r ig h t hand end o f S3 B x l (2 ) BamH I s h o r t term in u s from th e

l e f t hand end o f S4 t s l BamH I u

B ig e s te d w ith S s t I , e x c e p t f o r t r a c k 1$7 .5 # p o ly a c r y la m id e g e l .

C 1 . tsB BamH I pg.2 tsB BamH I s3 B xl (2 ) Bamli I s h o r t term in u s and la r g e r

lo n g term in u s4 B xl (2 ) Bamll I lo n g term in u s5 t s 6 BamH~~T~u6 t s 6 BamH I vw

B ig e s te d w ith S s t I ; 2 # a g a r o se g e l .

B 1 tsB BamH I pc2 TsB BamH I s3 jJ x l(2 j” BamH I s h o r t term in u s and la r g e r

lo n g term in u s '4 B x l (2 ) BamH I lo n g term in u s5 t s 6 . Bamii I. u6 t s 6 ’ Bamii I vw

B ig e s te d w itn Sma I ; 7 .5 # (u pp er) and 1 5 # ( lo w e r ) p o ly a c r y la m id e g e l s . * * -

H e s t r ic t io n fra g m en ts o f BamH I j o i n t (p a n e l A) and lo n g and s h o r t te r m in a l fra g m en ts (p a n e ls B-D) a re in d ic a t e d to th e l e f t (H SV -l) and r i g h t (HSV-2) o f each p a n e l . Xp d e n o te s th e te r m in a l S s t I or Sma I iragm en t o f L, and th e te r m in a l fragm en t o f S .

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Figure C 4 .17

D e ta ile d genome s tr u c tu r e s o f B x l(2 8 -1 ) su b c lon es

and o th e r recom binants: (a ) f ix e d in L, (b) f r e e in L.

and Ug have been sh o rten ed , and the c r o sso v e r in

fo r B x l(2 8 -1 ) su b c lo n es i s in the reg io n 0 .4 9 0 -

0^515. R ecta n g le s r e p r e se n t r e p e t i t iv e r e g io n s in the

c o n v e n tio n a l manner, b lack in d ic a t in g type 2 and

w h ite type 1 DNA. The a sequ en ces are shown on

a la r g e r s c a le fo r c l a r i t y . R e s t r ic t io n s i t e s

d e f in in g c r o ss o v e r s are shown a t the to p and bottom

o f th e diagram , and a ls o above (HSV-1) and below

(HSV-2) each r e p r e s e n ta t iv e genome (B BamH I ,

K Kpn I , R BcoR I , H Hpa I ) . C rosses in d ic a te

d e le te d seq u en ces and c r o s s -h a tc h in g shows r e g io n s

o f u n c e r ta in ty fo r c r o sso v e r s where g r e a te r than

300 bp. In some c a s e s c r o sso v e r s were determ ined

by f in e r e s t r i c t i o n mapping in the j o in t and

term in a l r e g io n s . Recombinant D x l(2 ) has two

h e te r o ty p ic a seq u en ces a t th e j o in t and so IRg

has been s h i f t e d r ig h tw a rd s.

aB x 1 (28-1-1)

8 x 1 ( 2 8 - 1 - 5 )

B x l (2 8 -1 - 1 4 )

bB x 1 (28-1-10)

B x 1 (28-1-11)

B x 1 ( 2 8 - 1 - 2 9 )

B * 1 ( 2 8 - 1 -9a)

B x 1 ( 2 8 -1 -95 )

B x l ( 2 8 - 1 - 2 2 )

B x 1 ( 3 1 - 2 )

B * 6 ( 1 7 - 6 )

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Figure 04.18

Upper p an el

Genome s tr u c tu r e s p o s s ib ly p r e se n t in B x l ( 2 8 - l ) ,

and th a t o f th e p u ta t iv e o r ig in a l recom binant B x l(28 ) from

which th ey a r o s e , B lack in d ic a t e s HSV-2 and w h ite HSV-1

seq u en ces.

Lower p an el

S tr u c tu r e s o f th e 4 o in t r e g io n s o f th r e e

recom binant genom es. B lack in d ic a t e s HSV-2 and w h ite HSV-1

seq u en ces, and c r o s s -h a tc h in g d e n o te s th e r e g io n o f

u n c e r ta in ty o f c r o sso v e r p o s i t io n . The a sequ en ces are

shown a s broader r e c ta n g le s and th e ju n c tio n between two

a seq u en ces by v e r t i c a l l i n e s ex ten d in g above and below them.

S iz e s o f n o v e l Smal fragm en ts c o n ta in in g th e c r o sso v e r s are

givien.

a Subclone 29 o f B x l(2 8 -1 ) (F igu re 04*10 p an el A

tr a c k 4; e n t ir e genome s tr u c tu r e in F igu re 0 4 * 1 7 ),

b B x l(2 ) (F igu re 04*16 p an el A tr a c k 3 ; e n t ir e

genome s tr u c tu r e in F igure 0 4 * 1 7 ),

c Subclone o f su b clon e 2 o f B x l(2 8 -1 ) (F igu re 04*16

p an el A tr a c k 4; e n t ir e genome s tr u c tu r e in

F igu re C 4 .21 , second typ e o f p ro g en y ).

putative original recombinant

I replication & t recombination?

28 bp-2a £

28 bp-2b = [

245 bp ±5c —m m

Figure 04*19

Genome s tr u c tu r e s o f su b c lo n es o f B x l(2 8 -1 ) used to

in v e s t ig a t e e x p r e ss io n o f IB 1 1 0 /1 1 8 .

Ujj and Ucj have been sh orten ed to em phasise d e t a i l s

o f the r e p e t i t iv e r e g io n s . R e s t r ic t io n s i t e s

w ith in r e p e t i t i v e r e g io n s are shown above the

genome fo r iiSV-1 and below fo r HSV-2 (B « BamH I ,

K « Kpn I , II *= Hpa I , H - KooB I ) . B lack d en otes

HSV-2 DNA and w h ite HSV-1 DBA, c r o s s -h a tc h in g

in d ic a t e s u n c e r ta in ty o f c r o sso v e r p o s i t io n where

g r e a te r than 3 0 0 bp, ana a c r o s s s i g n i f i e s

a e le te d se q u en ce s . The c r o sso v e r p o s i t io n in

i s between 0 .4 9 0 and 0 .515 f r a c t io n a l genome u n it s

fo r each su b c lo n e . The p o s i t io n s and d ir e c t io n o f

t r a n s c r ip t io n o f the genes cod in g fo r IB 110 and

IB l i b are shown (A. B aston , F. Rixon and J .B .

C lem ents, p e r so n a l com m unication).

I.E. 118 I.E. 110

8x1(28-1)

S1

S11

S29

S14

S 2 2

H

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1 1 B BB R R BB B j

BB B K

BB B KK B BB

F igure 0 4 .2 0

A utoradiograph o f IE p o ly p e p tid e s induced in HFL

c e l l s by HSV-1 tsB ( 1 1 ) , HSV-2 t s l (T 2),

recom binants B x l(3 1 -2 ) and B x l(2 8 -1 ) and su b c lo n es

(S) d er iv ed from B x l ( 2 8 - l ) . A lso in c lu d ed are

m o ck -in fec ted (MI) sam p les. IE p o ly p e p tid e s from

whole in f e c t e d c e l l s were sep ara ted on 5 - 1 2 ^

p olyacry lam id e g r a d ie n t g e l s ( la n e s 1 - 1 0 ) or

n u c lea r e x t i 'a c ts were made and sep arated on

7.5;^ p o lyacry lam id e g e l s ( la n e s 1 1 -2 1 ) . Numbers to

the l e f t o f la n e 1 and to th e r ig h t o f la n e 2 1 show

the apparent m olecu lar w e ig h ts (x 1CT3 ) o f HSV-1

IE p o ly p e p t id e s . Numbers between la n e s 10 and 11

show th e apparent m olecu lar w e ig h ts (x 1 0 *“^) o f

HSV-2 IE p o ly p e p t id e s .

Symbols are p la ced to th e l e f t o f the la n e to which

th ey r e f e r .

• HSV-1 polypex>tide

o HSV-2 p o ly p ep tid e

4 - a l t e r e d p o ly p ep tid e

I n is experim ent was perform ed by Dr H .S. Marsden.

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Figure C4.21

Genome s tr u c tu r e s o f th e progeny o f two su b c lo n es

o f B x l(2 8 -1 ) : (a ) su b clon e 2 9 , (b) su b clon e 2 .

Schem atic d e t a i l s are th e same a s in F igure

C4»17, e x ce p t th a t a seq u en ces are n o t em phasised .

rih e p a r e n ta l s tr u c tu r e s are shown, and the number

o f progeny w ith each r e s u l t in g genome s tr u c tu r e .

F in d ic a t e s f ix e d in L and N in d ic a t e s norm ally

in v e r t in g ( f r e e ) in L.

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10

3

3

1

ffi&ure C4.22

R e s t r ic t io n i^ r o f ile s o i in v iv o ^ P - la b e le d DNA from

RE4 (R) and some o f i t s su b c lo n es (numbered)

compared w ith HSV-1 (T l) and IISV-2 ( i ‘2) fo r Hind I I I

( l e f t ; 0.4/S agarose g e l) and HainH I ( r ig h t ; 1>

agarose g e l ) . HSV-1 r e s t r i c t i o n fragm ents are

in d ic a te d to the l e f t o f each p a n e l, and IISV-2

r e s t r i c t i o n fragm ents to the l e f t o f th e v e r t i c a l

l i n e .

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CO 1 1 1 111 II 1 ! 1 1 flfliLO 1 II III I I1 1 1 1 I

1 1 III 111 1 1 1 IBBCM 111 III I I 1 1 1 I I B

11 I I S II 1 1 1 warnCO IB I I1 1 I IBBCO 1 1 1 IB ■ 1 1 1 IBM

1 I 1 III B 1 1 1 1 IMCO *J 1 IB B 1 1 1 I B M

B O B B i 1 81- mcc 1 E 1 III I I1 1 1 i nCMH R B B 1 1 11K I I 1 IB III 1 1 m«oo 0 *•- o > - c c <0*0 -E c o

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Figure C4.23

H y b r id isa tio n o f s p e c i f i c probes to Southern b lo t s

o f HJS4 r e s t r i c t i o n d ig e s t s .

'i'he tra c k s are Hind I I I (upper h a lf o f F igu re) or

Bamli I (low er h a l f ) d ig e s t s o f HSV-1 ( T l) , HE4 (K)

or HSV-2 (T2) HNA tr a n s fe r r e d to n i t r o c e l lu lo s e from

agarose g e l s . A-C were tr a n sfe r r e d s im u lta n eo u sly

from the same g e l , and l ik e w is e H-E. The n i t r o c e l lu lo s e

f i l t e r s were c u t in to p o r t io n s c o n ta in in g th ree

tra ck s and h y b r id ised w ith n ic k tr a n s la te d BNA

a s f o l lo w s .

A HE4 HNA . . . -H recom binant p lasm id c o n ta in in g HSV-2 Hind I I I nC recom binant p lasm id c o n ta in in g HSV—2 HXnd I I I oH HE4 HNAS’ Ilind I I I TJ fragm ent i s o la t e d from HE4 HNA

( s l i g h t l y contam inated w ith RE4 Hind I I I x)

Those fragm ents which h y b r id ised to probes H, C and E

are in d ic a te d : HSV-1 fragm ents on the l e f t o f th e

tracic, HSV-2 on th e r ig h t . Fragments n o v e l to HE4

are in d ic a te d below th e band*. The in te r p r e ta t io n o f

the n o v e l fragm ents i s a s fo l lo w s ( s e e F igu re C 4 .2 6 ).

TJ Hind I I I or Hamll I fragm ent c o n ta in in g th e ju n c tio nbetween H3V-2 Hind I I I o (HSV-2 BarnH I f ) and HSV-2 Hind III~ b ( HSV-2~BamH I iT "

x Iiind I I I or Bamii I term in a l fragm ent o f Lgen era ted by tEe tr a n s p o s it io n

x x w ith an a d d it io n a l a sequence

j Hind I I I or Bamh I j o in t fragm ents c o n ta in in g x ,gen erated by the tr a n s p o s it io n

TI R T2

S I

§ c D ETl R T2 TI R T2 Tl R T2 TI R T2

a bed H t -

>n

ATl R T2

SB

X

Xn

TJ

o

Tl R T2 Tl R T2 Tl R T2 Tl

I 5 i

ER T2

TJ

cr

f f

Figure C4.24

Location of the inserted DNA in RE4.

A M pBR322 H a e l l l markers1 HSV-2 BamHI i d ig e s te d w ith Hhal2 RE4 BamHT U n d ig e s te d w ith HEaT3 RE4 BlndllTZBamHI TJ d ig e s te T ”w ith Hhal

. 4 HSV-2 H in d III-o - d ig e s te d w ith Hhal5 HSV-2 BamHI i H ig ested w ith AIuTT6 RE4 BamHT. U n d ig e s te d w ith Alul7 RE4 Hind I IlZBamHI TJ d ig e s te d -w ith Alul8 HSV-2-Hindl l l o- d ig e s te d w ith A lul - - -

7 . 57* (upper) and 1 5 7 * (lo w er) po lyacry lam id e g e l s .

B 1 HSV-2 BamHI 1 d ig e s te d w ith Smal2 HE4 BamHI U n d ig e s te d w ith SmaT3 HE4 HindIIl7BamHI TJ d ig e s te d w ith Smal4 HSV-2 .BindI I I o, d ig e s te d w ith Smal .7 M pBR322’~nTnfI markers

2 $ a g arose g e l .

Marker s i z e s are in d ic a te d in bp.

TJ d e n o te s a r e s t r i c t i o n fragm ent c o n ta in in g th e ju n c tio n Eetween norm al HSV-2 and in s e r te d HSV-2 seq u en ces.The lo c a t io n s o f BamHI TJ and th e double d ig e s t fragm ent HindI I l 7 BamHI TJ are. shown in F igu re C4#26.

A M 1 2 3 4 5 6 7 8 M B 1 2 3 4 M

•—« H —

■ - #

- 4

-1631

• - 5 0 6

- 3 9 6- 3 4 4

• - 2 9 8

m -220

Figure 0 4 .2 5

Structures of the joints and termini o f RE4.

A 1 tsB BamH I k2 RE4 BamH I major j o in t3 t g l BamH I g

D ig e sted w ith S s t I ; 2/« agarose g e l .

B 1 tsB BamH I c[* 2 RE4 BamH I major sh o r t term inu s, i s o la t e d

from type 1 EcoR I &3 RE4 BamH I major sh o r t term in u s, i s o la t e d

from type 1 Hind I I I m

D ig e sted w ith S s t I ; 2)* agarose g e l .

C 1 tsB BamH I k“ 2 1124 BamH I major j o in t

3 t s l BamH I g4 t s l BamH I r3 RE4 BamH I n o v e l lo n g term inus6 RE4 Hind I I I n o v e l lo n g term inus

D ig e sted w ith Sma I ; 7#5‘* p o lyacry lam id e g e l .

D 1 t s B Bamii I k2 RE4 BamH I major j o in t3 t s l Bamii I g

D ig e sted w ith Sma I ; 15/* p o lyacry lam id e g e l .

E 1 RE4 BamH I type 1 lo n g term inus 2 tsB Damn I . s

D ig ested w ith Sma I ; 7 #5/* p o lyacry lam id e g e l .

B 1 tsB BamH I c[2 RE4 BamH I major sn o r t term inus

D ig e sted w ith Sma I ; 7 .5 ^ p o lyacry lam id e g e l .

S s t I or Sma I frag iaen ts o f the j o in t or term in a l fragm ents o f IiSV-1 ( l e f t ) and HSV-2 ( r ig h t ) are in d ic a te d . X d en o tes a genome term in a l fragm ent.

CM

i i i i i i< 0 0 LU U_ (J I i i

o a x

cm I 4* 1— | M 1 •UJ| 6 Q

S.COO)9^'_I 1 1« » * •« «

i i iI ---- 5CD KL

--1

2

N-

w mqi ^ z oq. a Icc

r - cm co in CDI -7 8 9 10

11

t f im^ I f l H H i B Mco ' 1 1cm t-

oi < coo g ii II 1 1I CD “ “5

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CM 0 0 ^A A 1

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figure C4.26

Genome structure of RE4.

■BamHI fragm ents p r e se n t in RE4 DHA are shown a t

th e top o f th e F ig u r e . B lach in d ic a t e s HSV-2 and w h ite

USV-1 seq u en ce . R egions o f u n c e r ta in ty o f c r o sso v e r

lo c a t io n are shown by h o r iz o n ta l h a tch in g and th e in s e r te d

HSV-2 sequence from i s denoted by c r o s s -h a tc h in g .

The n atu re o f th e a seq u en ces (HSV-1 or HSV-2: 1 or 2)

i s shown in s id e th e s t a r s . RE4 i s f ix e d in L and S in the

1,3 arrangem ent a s i l l u s t r a t e d .

The low er p a r t o f th e F igu re shows HSV-2 Hind iI I

and Hamill fragm en ts which are p r e se n t in th e r e g io n o f th e

in s e r t io n , v e r t i c a l arrows in d ic a t in g r e s t r i c t i o n s i t e s .

S iz e s in bp o f Sm al. Alul and Hhal fragm ents c o n ta in in g

the ju n c tio n between normal and in s e r te d ( 11 tra n sp o sed ” )

HSV-2 sequ en ces arc g iv e n . To th e r ig h t in p a re n th e se s

are g iv e n s i z e s in bp o f fragm ents produced by d ig e s t io n

o f th e H in d i! I / Bamiil fragm ent c o n ta in in g th e ju n c tio n

which were a ls o produced from HSV-2 BamHI i .

O)

'CO

Qg0&0CO

sC/>x“O0coa</>cCO

0)0 c 0) 3 O ’ 0) CO

CM

1x"coEoc

I3

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O’0CO

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XECO

GO

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(/)3<

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h

Figure 04.27 (4 pages)

Models for the inversion of L and S in HSV PNA.

Model 1 shows s e p a r a tio n o f 1 and S, in l in e a r

or c ir c u la r form , and t h e ir su bsequent recom b in ation in the

a seq u en ces (Hayward e t a l . , 1975b; Share and Summers, 1 9 7 7 ) .

Model 2 shows in te r m o le c u la r r ecom bination o f

l in e a r or c ir c u la r m o lec u le s in a r e p e a t sequence to

p ro d u ce 'th e o th e r arrangem ents a f t e r a p p ro p ria te c le a v a g e

e v e n ts (Share and Summers, 1 9 7 7 ) .

Model 3A shows in tr a m o le c u la r recom b in ation

a f t e r MA s y n th e s is u s in g a s in g le stran d o f th e genome

a s tem p la te (Share and Summers, 1 9 7 7 ) .

Model 3B shows in tr a m o le c u la r recom b in ation

in l in e a r or c ir c u la r form in a r e p e a t sequence

(S h e ld r ic h and B e r th e lo t , 19745 Share and Summers, 1977;

Sm iley e t a Z ,, 1 9 8 0 ) .

Model 30 shows in tr a m o le c u la r recom b in ation

in v o lv in g r e p a ir o f a seq u en ces (Jacob and Roizman, 1979;

Roizman, 1 9 7 9 ) . B r ie f ly , the model in v o lv e s c ir c u la r i s a t io n

o f th e l in e a r DNA m olecu le by th e a c t io n o f an ex o n u c lea se

fo llo w e d by l i g a t i o n o f com plem entary s in g le stranded

te r m in i, DNA i s r e p l ic a t e d by a r o l l i n g c i r c l e mechanism

to g en era te con catem ers, which are c le a v ed in a lt e r n a te

j o in t r e g io n s to produce u n i t - 1 ength m o lecu les la c h in g

an a sequence a t one term in u s. The m iss in g a sequence

i s reg en era ted by "cop y in g 11 from the j o in t r e g io n .

At t h i s p o in t , n ic k in g a t th e j o in t a sequence and

branch m ig ra tio n c a u se s in v e r s io n o f th e segm ent to

produce th e P, Ig and 1 ^ arrangem ents. The Igj,

arrangem ent i s produced"by a second round o f ”

r e p l i c a t io n o f Ig or 1^, w ith con cu rren t in v e r s io n .

1

I— I-

I

I

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C Z T J-M 3

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o

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5L

Comparison o f the e f f e c t s upon m aturation o f

u n it - le n g th genomes o f two mechanisms fo r g e n e ra tio n

o f c ir c u la r HSV DNA.

B lun t-en d l ig a t io n produces a c i r c l e w ith d is s im ila r

j o in t s , whereas complem entqry-end l i g a t i o n produces a

c i r c l e w ith id e n t i c a l j o i n t s . I n i t i a l progeny m o lecu les

may in v e r t n e i th e r , e i t h e r or both segm ents to produce

the fo u r c ir c u la r arrangem ents. In t h i s diagram model 3B

fo r s i t e - s p e c i f i c in tr a m o le c u la r recom b in ation h as been

invoked (F igu re C 4 .2 7 ). Four arrangem ents o f concatem ers

are then gen erated by r o l l i n g c i r c l e DHA s y n t h e s is .

C leavage may occu r a t any j o in t o f concatem ers

r e s u l t in g from com plem entary-end l i g a t i o n , so th a t each

concatem er arrangem ent produces two l in e a r genome

arrangem ents. C leavage cou ld r e s u l t frpm a s in g le

strand n ic k a t the ends o f the a seq u en ce , fo llo w e d by

r e p a ir o f th e s in g le stranded a sequence a t the term in i

o f u n it - le n g th m o le c u le s . I t i s n e c e ssa r y f o r o n ly one

segment to in v e r t to produce the fo u r genome arrangem ents,

and iso m e r isa t io n i s a fu n c t io n o f both in v e r s io n and

c le a v a g e .

C on verse ly , c le a v a g e may occur o n ly a t a l t e r n a te

j o in t s o f concatem ers r e s u l t in g from b lu n t-en d l i g a t i o n ,

and each concatem er produces o n ly one l in e a r genome

arrangem ent. C leavage in t h i s ca se cou ld r e s u l t frpm a

double stranded cu t between two a seq u en ces ( i . e . a t th e

j o in t which i s n o t c le a v ed in model 3C o f F igure C 4*27).

I t i s n e c essa ry fo r both segm ents to in v e r t to g en era te

the fou r arrangem ents. I so m e r isa t io n i s a fu n c t io n

only o f in v e r s io n . A lt e r n a t iv e ly , th e c lea v a g e

mechanism may r e c o g n ise both ty p e s p f j o i n t , and in t h i s

ca se the two m odels fo r c ir c u la r i s a t io n have id e n t i c a l

e f f e c t s upon m aturation ( i . e . th a t o f com plem entary-end l ig a t io n

ab

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l l E J E E E N C i S

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